EP2125893A2 - Optimierte cd40-antikörper und anwendungsverfahren dafür - Google Patents
Optimierte cd40-antikörper und anwendungsverfahren dafürInfo
- Publication number
- EP2125893A2 EP2125893A2 EP08728147A EP08728147A EP2125893A2 EP 2125893 A2 EP2125893 A2 EP 2125893A2 EP 08728147 A EP08728147 A EP 08728147A EP 08728147 A EP08728147 A EP 08728147A EP 2125893 A2 EP2125893 A2 EP 2125893A2
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- EP
- European Patent Office
- Prior art keywords
- antibody
- amino acid
- antibodies
- acid sequence
- seq
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/08—Antiallergic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/72—Increased effector function due to an Fc-modification
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
Definitions
- the present invention relates to optimized proteins that target CD40, and their application, particularly for therapeutic purposes.
- B cells are lymphocytes that play a large role in the humoral immune response. They are produced in the bone marrow of most mammals, and represent 5-15% of the circulating lymphoid pool. The principal function of B cells is to make antibodies against various antigens, and are an essential component of the adaptive immune system. [04] The human body makes millions of different types of B cells each day that circulate in the blood and lymph performing the role of immune surveillence. B cells, also referred to as B lymphocytes, do not produce antibodies until they become fully activated. Each B cell has a unique receptor protein (referred to as the B cell receptor (BCR)) on its surface that will bind to one particular antigen.
- BCR B cell receptor
- the BCR is a membrane-bound immunoglobulin, and it is this molecule that allows the distinction of B cells from other types of lymphocytes, as well as being the main receptor involved in B-cell activation.
- a B cell encounters its cognate antigen and receives an additional signal from a T helper cell, it can further differentiate into the various types of B cells listed below.
- the B cell may either become one of these cell types directly or it may undergo an intermediate differentiation step, the germinal center reaction, where the B cell will hypermutate the variable region of its immunoglobulin gene and possibly undergo class switching.
- B-cell development occurs through several stages, each stage representing a change in the genome content at the antibody loci.
- B-cell development includes Progenitor B cells, Early Pro-B cells, Late Pro-B cells, Large Pre-B cells, Small Pre-B cells, Immature B cells, and Mature B cells.
- Mature B cells can be divided into four major types: [07] B-1 cells express CD5, a marker usually found on T cells. B-1 cells also express IgM in greater quantities than IgG. They secrete natural low affinity polyreactive antibodies found in the serum and often have specificities directed toward self-antigens, and common bacterial polysaccharides. B-1 cells are present in low numbers in the lymph nodes and spleen and are instead found predominantly in the peritoneal and pleural cavities. [08] B-2 cells are the conventional B cells to which most texts refer. They reside in bone marrow, spleen, and lymph nodes. They are short-lived, and when triggered by antigens may differentiate into IgG-producing memory B cells. In the course of these antibody responses IgG may undergo substantial affinity maturation.
- Plasma B cells are large B cells that have been exposed to antigen and produce and secrete large amounts of antibodies, which assist in the destruction of microbes by binding and facilitating targeting by phagocytes, as well as activation of the complement system. Plasma cells are sometimes referred to as antibody factories.
- Memory B cells are formed from activated B cells that are specific to the antigen encountered during the primary immune response. These cells live for a long time, and can respond quickly following a second exposure to the same antigen.
- a B cell fails in any step of the maturation process, it will die by a mechanism called apoptosis. If it recognizes self-antigen during the maturation process, the B cell will become suppressed (known as anergy) or undergo apoptosis. B cells are continuously produced in the bone marrow, but only a small portion of newly made B cells survive to participate in the long-lived peripheral B-cell pool.
- B lymphocytes play a broader role in immune responses and are not merely the passive recipients of signals that result in differentiation into antibody-producing plasma cells.
- B cells have also been found to regulate antigen presenting cells (APCs) and T-cell functions, produce cytokines, and express receptor/ligand pairs that previously had been thought to be restricted to other cell types.
- APCs antigen presenting cells
- T-cell functions produce cytokines, and express receptor/ligand pairs that previously had been thought to be restricted to other cell types.
- the present invention is directed to antibodies and methods of using the same.
- the antibodies include a variant Fc region.
- the present invention is further directed to methods of using the antibodies in various disease indications.
- the present invention is directed to an antibody that binds CD40, wherein said antibody comprises at least one modification in the constant region relative to a parent antibody.
- the antibody of the invention binds with altered affinity to an Fc receptor or alters effector function as compared to the parent antibody.
- the modification is an amino acid.
- the modification can be at a position selected from the group consisting of 221 , 222, 223, 224, 225, 227, 228, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 255, 258, 260, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271 , 272, 273, 274, 275, 276, 278, 280, 281 , 282, 283, 284, 285, 286, 288, 290, 291 , 292, 293, 294, 295, 296, 297, 298, 299, 300, 301 , 302, 303, 304, 305, 313, 317, 318, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331 , 332, 333, 334, 3
- the amino acid modification can be a substitution selected from the group consisting of 221 K, 221 Y, 222E, 222Y, 223E 1 223K, 224E, 224Y, 225E, 225K, 225W, 227E, 227G, 227K, 227Y, 228E, 228G, 228K, 228Y, 230A, 230E, 230G, 230Y, 231 E, 231 G, 231 K, 231 P, 231 Y, 232E, 232G, 232K, 232Y, 233A, 233D, 233F, 233G, 233H, 233I, 233K, 233L, 233M, 233N, 233Q, 233R, 233S, 233T, 233V, 233W, 233Y, 234A, 234D, 234E, 234F, 234G, 234H, 234!, 234K, 234M, 234N,
- the amino acid modification can be at a position selected from the group consisting of 221 , 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241 , 243, 245, 246, 247, 249, 255, 258, 260, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271 , 272, 274, 275, 276, 278, 280, 281 , 282, 283, 284, 285, 286, 288, 290, 291 , 292, 293, 294, 295, 296, 297, 298, 299, 300, 301 , 302, 303, 304, 305, 313, 317, 318, 320, 322, 324, 325, 326, 327, 328, 329, 330, 331 , 332, 333, 334, 335, 336, and 337.
- the substitution can be selected from the group consisting of 221 K 1 222Y, 223E, 223K, 224E, 224Y 1 225E 1 225W, 227E, 227G, 227K, 227Y, 228E 1 228G 1 228K, 228Y, 230A, 230E, 230G, 230Y, 231 E, 231 G, 231 K, 231 P 1 231Y 1 232E, 232G 1 232K 1 232Y, 233A, 233F, 233H, 2331, 233K, 233L, 233M, 233N, 233Q, 233R, 233S, 233T, 233V, 233W, 233Y, 234D, 234E 1 234F, 234G, 234H 1 2341, 234K, 234M, 234N, 234P 1 234Q 1 234R, 234S, 234T, 234W, 234Y, 235
- the modification is at a position selected from the group consisting of 221 , 222, 223, 224, 225, 228, 230, 231 , 232, 240, 244, 245, 247, 262, 263, 266, 271 , 273, 275, 281 , 284, 291 , 299, 302, 304, 313, 323, 325, 328, 332, 336, wherein the positional numbering is according to the EU index.
- the modification is selected from the group consisting of 221 K, 221 Y, 222E, 222Y, 223E, 223K, 224E, 224Y, 225E, 225K, 225W, 228E, 228G, 228K, 228Y, 230A, 230E, 230G, 230Y, 231 E, 231 G, 231 K, 231 P, 231Y, 232E, 232G, 232K, 232Y, 240A, 240I, 240M, 240T, 244H, 245A, 247G, 247V, 262A, 262E, 262F, 262I, 262T, 263A, 263I, 263M, 263T, 266A, 266I, 266M, 266T, 271 A, 271 D, 271 E, 271 F, 271G, 271 H, 2711, 271K, 271 L, 271 M, 271 N, 2
- the antibody can further include a second amino acid modification at a position selected from the group consisting of 221 , 222, 223, 224, 225, 227, 228, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241 , 243, 244, 245, 246, 247, 249, 255, 258, 260, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271 , 272, 273, 274, 275, 276, 278, 280, 281, 282, 283, 284, 285, 286, 288, 290, 291 , 292, 293, 294, 295, 296, 297, 298, 299, 300, 301 , 302, 303, 304, 305, 313, 317, 318, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331 , 332, 333, 334, 335, 336
- the second amino acid modification can be a substitution selected from the group consisting of 221 K, 221Y, 222E, 222Y, 223E, 223K, 224E, 224Y 1 225E, 225K, 225W, 227E, 227G, 227K, 227Y, 228E, 228G, 228K, 228Y, 230A, 230E, 230G, 230Y, 231 E, 231 G, 231 K, 231 P, 231 Y, 232E, 232G, 232K, 232Y, 233A, 233D, 233F, 233G, 233H, 233I, 233K, 233L, 233M, 233N, 233Q, 233R, 233S, 233T, 233V, 233W, 233Y, 234A, 234D, 234E, 234F, 234G, 234H, 234I, 234K, 234M, 234N,
- the amino acid modification is 332E.
- the second amino acid modification can be selected from the group consisting of: 236A, 239D, 332E 1 268D, 268E, 330Y, and 330L. In certain embodiments, the second amino acid modification is 239D.
- the modification is a glycoform modification that reduces the level of fucose relative to the parent antibody.
- the invention is directed to a composition including a plurality of glycosylated antibodies, wherein about 80-100% of the glycosylated antibodies in the composition comprise a mature core carbohydrate structure which lacks fucose.
- the antibody reduces binding to Fc ⁇ Rllb as compared to the parent anti-CD40 antibody.
- the invention is directed to an antibody that binds CD40, includes a heavy chain and/or a light chain, and has an increased affinity to the Fc ⁇ Rllla receptor as compared to the parent antibody.
- the heavy chain has a CDR1 comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 22, and 28, a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 23, and 29, and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 24, and 30.
- the light chain has a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 25, and 31 , a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 26, and 32, and a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 21 , 27, and 33.
- the antibody has a variable heavy chain sequence selected from the group consisting of SEQ ID NOs: 10, 12, 14, and 16-19 and/or a variable light chain sequence selected from the group consisting of SEQ ID NOs: 11 , 13, 15 and 20-22.
- the invention is directed to a nucleic acid sequence encoding any of the antibodies disclosed herein.
- the invention is directed to a method of treating a B-cell related disease by administering an antibody according to claim 1.
- the disease is selected from non-Hodgkin's lymphomas (NHL), chronic lymphocytic leukemia (CLL), B- cell acute lymphoblastic leukemia/lymphoma (B-ALL), mantle cell lymphoma (MCL), and multiple myeloma (MM).
- the disease is an autoimmune disease, such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE or lupus), multiple sclerosis, Sjogren's syndrome, and idiopathic thrombocytopenia purpura (ITP).
- RA rheumatoid arthritis
- SLE or lupus systemic lupus erythematosus
- multiple sclerosis multiple sclerosis
- Sjogren's syndrome idiopathic thrombocytopenia purpura
- ITP idiopathic thrombocytopenia purpura
- the invention is directed to a composition comprising an antibody described herein and an acceptable carrier.
- the invention is directed toward an antibody that binds CD40, wherein the antibody comprises a means for optimizing effector function.
- the means allows for antibody binding with increased affinity to the Fc ⁇ Rllla receptor as compared to the parent antibody.
- the means is an amino acid modification.
- the means is a positional means for optimizing effector function, e.g., modification of an amino acid at one or more of the following heavy chain constant region positions: 221 , 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241 , 243, 244, 245, 246, 247, 249, 255, 258, 260, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271 , 272, 273, 274, 275, 276, 278, 280, 281, 282, 283, 284, 285, 286, 288, 290, 291 , 292, 293, 294, 295, 296, 297, 298, 299, 300, 301 , 302, 303, 304, 305, 313, 317, 318, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330,
- the means is a substitutional means for optimizing effector function, e.g., one or more of the following amino acid substitutions in the heavy chain constant region positions: 221 K, 221 Y, 222E, 222Y, 223E, 223K, 224E, 224Y, 225E, 225K, 225W, 227E, 227G, 227K, 227Y, 228E, 228G, 228K, 228Y, 230A, 230E, 230G, 230Y, 231 E, 231 G, 231 K, 231 P, 231 Y, 232E, 232G, 232K, 232Y, 233A, 233D, 233F, 233G, 233H, 233I, 233K, 233L, 233M, 233N, 233Q, 233R, 233S, 233T, 233V, 233W, 233Y, 234A, 234D, 234E, 2
- the means for optimizing effector function is a positional means, e.g., modification of an amino acid at one or more of the following positions: 221, 222, 223, 224, 225, 228, 230, 231 , 232, 240, 244, 245, 247, 262, 263, 266, 271 , 273, 275, 281 , 284, 291 , 299, 302, 304, 313, 323, 325, 328, 332, 336, wherein the positional numbering is according to the EU index.
- positional means e.g., modification of an amino acid at one or more of the following positions: 221, 222, 223, 224, 225, 228, 230, 231 , 232, 240, 244, 245, 247, 262, 263, 266, 271 , 273, 275, 281 , 284, 291 , 299, 302, 304, 313, 323, 325, 328, 332, 336, wherein the positional numbering is according to the EU index.
- the means for optimizing effector function is a substitutional means, e.g., one or more of the following substitutions: 221 K, 221 Y, 222E, 222Y, 223E, 223K, 224E, 224Y, 225E, 225K, 225W, 228E, 228G, 228K, 228Y, 230A, 230E, 230G, 230Y, 231 E, 231 G, 231 K, 231 P, 231 Y, 232E, 232G, 232K, 232Y, 240A, 240I, 240M, 240T, 244H, 245A, 247G, 247V, 262A, 262E, 262F, 262I, 262T, 263A, 263I, 263M, 263T, 266A, 266I, 266M, 266T, 271A, 271 D, 271 E, 271 F, 271G, 271
- the means for optimizing effector function includes a modification at a second amino acid, e.g., at a position selected from the group consisting of 221 , 222, 223, 224, 225, 227, 228, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241 , 243, 244, 245, 246, 247, 249, 255, 258, 260, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271 , 272, 273, 274, 275, 276, 278, 280, 281 , 282, 283, 284, 285, 286, 288, 290, 291 , 292, 293, 294, 295, 296, 297, 298, 299, 300, 301 , 302, 303, 304, 305, 313, 317, 318, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330
- the means for optimizing effector function may include a substitution at a second amino acid, e.g., one or more of the following substitutions: 221 K, 221 Y, 222E, 222Y, 223E, 223K, 224E, 224Y, 225E, 225K, 225W, 227E, 227G, 227K, 227Y, 228E, 228G, 228K, 228Y, 230A, 230E, 230G, 230Y, 231 E, 231 G, 231 K, 231 P, 231 Y, 232E, 232G, 232K, 232Y, 233A, 233D, 233F, 233G, 233H, 233I, 233K, 233L, 233M, 233N, 233Q, 233R, 233S, 233T, 233V, 233W, 233Y, 234A, 234D, 234E, 234F, 234G, 2
- the means for optimizing effector function is the amino acid modification 332E.
- the means for optimizing effector function is the amino acid modification 332E and one or more of the following amino acid modifications: 236A, 239D, 332E, 268D, 268E, 330Y, and 330L, e.g., 239D.
- an antibody of the invention has a reduced level of fucose relative to the parent antibody.
- a composition of the invention may comprise a plurality of glycosylated antibodies, wherein about 80-100% of the glycosylated antibodies have a reduced level of fucose.
- an antibody of the invention comprises a means to reduce binding to Fc ⁇ Rllb as compared to the parent anti-CD40 antibody.
- Figure 1 Sequences of the natural antibody constant regions, including the kappa constant light chain (SEQ ID NO:1), and the gamma constant heavy chains for IgGI , lgG2, lgG3, and lgG4 (SEQ ID NOs:2-5, respectively). Also provided in Figure 1 b is the sequence of a Hybrid IgG constant chain (SEQ ID NO:6), and a Hybrid IgG constant chain comprising the substitutions 239D and I332E (SEQ ID NO:7).
- FIG. 1c depicts the alignment of the amino acid sequences of the human IgG immunoglobulins IgGI , lgG2, lgG3, and lgG4.
- FIG. 1c provides the sequences of the CH1 (C ⁇ 1) domain, hinge domain, CH2 (C ⁇ 2) domain and CH3 (CJ ⁇ 3) domain. Positions are numbered according to the EU index of the IgGI sequence, and differences between IgGI and the other immunoglobulins lgG2, lgG3, and lgG4 are shown in grey. Polymorphisms exist at a number of positions (Kim et al., 2001 , J. MoI. Evol. 54:1-9, incorporated herein by reference in its entirety), and thus slight differences between the presented sequences and sequences in the prior art may exist. The possible beginnings of the Fc region are labeled, defined herein as either EU position 226 or 230.
- Figure 2 The common haplotypes of the gamma chain of human IgGI ( Figure 2a) and lgG2 ( Figure 2b) showing the positions and the relevant amino acid substitutions.
- Figure 3 Embodiments of receptor binding profiles that include increases to, reductions to, or no effect on the binding to various receptors, where such changes may be beneficial in certain contexts.
- Figure 4 Amino acid sequences of the CD40 antigen targeted by the antibodies of the invention.
- Figure 4 provides the sequence of both isoforms of Homo sapiens CD40.
- Figure 5. Sequences encoding the murine anti-CD40 antibody variable regions.
- Figure 5 provides the light and heavy chain variable region sequences of the anti-CD40 antibodies S2C6, G28-5, and 5D12, respectively. CDRs are underlined.
- FIG. 5b-5d Also shown in Figure 5b-5d are heavy chain variable (VH) and light chain variable (VL) CDR sequences of the anti-CD40 antibodies S2C6 ( Figure 5b; SEQ ID NOs: 16-21 ), G28-5 ( Figure 5c (SEQ ID NOs:22-27), and 5D12 ( Figure 5d; SEQ ID NOs:28-33).
- Figure 6. ADCC assay comparing WT IgGI , Hybrid S239D/I332E (effector function enhanced) anti-CD40 antibodies, and the anti-CD20 antibody rituximab on the Burkitt's
- FIG. 7 Platelet activation assay with anti-CD40 antibody S2C6. Samples included in the assay were S2C6 IgGI , S2C6 Hybrid S239D/I332E, positive control antibody IV.3 (mouse anti-human IgG FcgRlla specific antibody), and isotype control antibody. [41] Figure 8. Anti-proliferation assay of anti-CD40 antibodies on the Burkitt's Lymphoma cell line HS-Sultan. Samples included in the assay were 5D12 IgGI , S2C6 IgGI , and human IgG with and without CD40 ligand and cells only were used as controls. [42]
- B-cell disorders Because of their critical role in regulating the immune system, disregulation of B cells is associated with a variety of disorders. B-cell disorders, also referred to herein as B-cell related diseases, are divided into excessive or uncontrolled proliferation (lymphomas, leukemias), and defects of B-cell development/immunoglobulin production (immunodeficiencies). The majority (80%) of lymphoma cases are of B-cell origin. These include non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), and autoimmune related diseases.
- NHL non-Hodgkin's lymphoma
- ALL acute lymphoblastic leukemia
- NHL is a heterogeneous malignancy originating from lymphocytes. In the United States (U.S.), the incidence is estimated at 65,000/year with mortality of approximately 20,000 (American Cancer Society, 2006; and SEER Cancer Statistics Review). The disease can occur in all ages, the usual onset begins in adults over 40 years of age, with the incidence increasing with age. NHL is characterized by a clonal proliferation of lymphocytes that accumulate in the lymph nodes, blood, bone marrow and spleen, although any major organ may be involved. [45] The diagnosis and histologic characterization of NHL is made using a combination of morphologic and immunophenotype criteria.
- the current classification system used by pathologists and clinicians is the World Health Organization (WHO) Classification of Tumours, which organizes NHL into precursor and mature B-cell or T-cell neoplasms.
- WHO World Health Organization
- the PDQ is currently dividing NHL as indolent or aggressive for entry into clinical trials.
- the present document will also use a similar division.
- the indolent NHL group is comprised primarily of follicular subtypes, small lymphocytic lymphoma, MALT, and marginal zone; indolent encompasses approximately 50% of newly diagnosed B-cell NHL patients.
- Aggressive NHL includes patients with histologic diagnoses of primarily diffuse large B cell (40% of all newly diagnosed patients have diffuse large cell), Burkitt's, and mantle cell.
- NHL The clinical course of NHL is highly variable. A major determinant of clinical course is the histologic subtype. Most indolent types of NHL are considered to be incurable diseases. Patients respond initially to either chemotherapy or antibody therapy and most will relapse. Studies to date have not demonstrated an improvement in survival with early intervention. In asymptomatic patients, it is acceptable to "watch and wait” until the patient becomes symptomatic or the disease pace appears to be accelerating. Over time, the disease may transform to a more aggressive histology. The median survival is 8 to 10 years, and indolent patients often receive 3 or more treatments during the treatment phase of their disease. Initial treatment of the symptomatic indolent NHL patient historically has been combination chemotherapy.
- the most commonly used agents include: cyclophosphamide, vincristine and prednisone (CVP); cyclophosphamide, adriamycin, vincristine, prednisone (CHOP); or the purine analog, fludarabine.
- CVP vincristine and prednisone
- CVP cyclophosphamide, adriamycin, vincristine, prednisone
- fludarabine purine analog
- the discovery and clinical use of the anti-CD20 antibody, rituximab has provided significant improvements in response and survival rate.
- the current standard of care for most patients is rituximab + CHOP (R-CHOP) or rituximab + CVP (R-CVP).
- CLL chronic lymphocytic leukemia
- CLL chronic lymphoid leukemia
- CLL is an incurable disease but progresses slowly in most cases. Many people with CLL lead normal and active lives for many years. Because of its slow onset, early-stage CLL is generally not treated since it is believed that early CLL intervention does not improve survival time or quality of life. Instead, the condition is monitored over time. Initial CLL treatments vary depending on the exact diagnosis and the progression of the disease. There are dozens of agents used for CLL therapy.
- fludarabine Although the purine analogue fludarabine was shown to give superior response rates than chlorambucil as primary therapy, there is no evidence that early use of fludarabine improves overall survival.
- Combination chemotherapy regimens such as fludarabine with cyclophosphamide, FCR (fludarabine, cyclophosphamide and rituximab) and CHOP are effective in both newly-diagnosed and relapsed CLL. Allogeneic bone marrow (stem cell) transplantation is rarely used as a first-line treatment for CLL due to its risk.
- Refractory CLL is a disease that no longer responds favorably to treatment. In this case more aggressive therapies, including bone marrow (stem cell) transplantation, are considered.
- the monoclonal antibody alemtuzumab, directed against CD52, may be used in patients with refractory, bone marrow-based disease.
- Another type of leukemia is acute lymphoblastic leukemia (ALL), also known as acute lymphocytic leukemia. ALL is characterized by the overproduction and continuous multiplication of malignant and immature white blood cells (also known as lymphoblasts) in the bone marrow.
- ALL acute lymphoblastic leukemia
- ALL is characterized by the overproduction and continuous multiplication of malignant and immature white blood cells (also known as lymphoblasts) in the bone marrow.
- ALL is most common in childhood with a peak incidence of 4-5 years of age. Children of age 12-16 die more easily from it than those of other ages. Currently, at least 80% of childhood ALL are considered curable. Under 4,000 cases are diagnosed every year and the mortality rate is almost 1 ,500 a year (American Cancer Society, 2006; and SEER Cancer Statistics Review).
- MM Multiple myeloma
- MM is a B-cell malignancy with terminally differentiated plasma cells. The disease subsequently attacks bone and bone marrow, which results in multiple tumors and lesions throughout the skeletal system. It is the second most common hematologic malignancy in the United States, afflicting approximately 55,000 people there alone. The annual incidence rate for multiple myeloma is 3 to 4 cases per 100,000 people, making it the most common bone tumor cancer in adults.
- Current treatment protocols which include a combination of chemotherapeutic agents yield a complete remission rate of only about 5%, with a median survival of approximately 36-48 months from the time of diagnosis. Treatment regimens are limited by a low cell proliferation rate and the development of multidrug resistance.
- autoimmune diseases include, for example, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE or lupus), multiple sclerosis (MS), Sjogren's syndrome, and idiopathic thrombocytopenia purpura (ITP).
- RA rheumatoid arthritis
- SLE or lupus systemic lupus erythematosus
- MS multiple sclerosis
- Sjogren's syndrome idiopathic thrombocytopenia purpura
- Autoantibodies are produced by terminally differentiated plasma cells that are derived from naive or memory B cells. Furthermore, B cells can have other effects on autoimmune pathology, as antigen-presenting cells (APCs) that can interact with and stimulate helper T cells, further stimulating the cycle of anti-self immune response. Depletion of B cells can have direct impact on the production of autoantibodies. Indeed, treatment of RA and SLE with B-cell depletion therapies such as rituximab has been demonstrated to have clinical benefit for both disease classes (Edwards & Cambridge, Nat. Rev. Immunol.
- Monoclonal antibodies are a class of therapeutic proteins that may be used to treat B-cell disorders.
- a number of favorable properties of antibodies including but not limited to specificity for target, ability to mediate immune effector mechanisms, and long half-life in serum, make antibodies powerful therapeutics.
- the present invention describes antibodies against the B-cell and solid tumor antigen CD40.
- CD40 antigen is a cell surface glycoprotein which belongs to the tumor necrosis factor receptor (TNF-R) family and is expressed on the surface of all mature B cells, most mature B-cell malignancies, some early B-cell acute lymphocytic leukemias, and -70% of all solid tumors. CD40 is also expressed on dendritic cells, monocytes, endothelial cells, epithelial cells, fibroblasts, smooth muscle cells, and many human solid tumors, including melanoma and carcinomas. Signaling via CD40 activates antigen-presenting cells, including B-cells and dendritic cells.
- TNF-R tumor necrosis factor receptor
- CD154 which is the natural ligand of CD40, is expressed on the surface of activated T-lymphocytes and provides a large component of T-cell triggering for immune responses. Consequently, CD40 agonists trigger immune responses against many tumor-associated antigens. Because of these properties, and its expression on many B-cell malignancies and solid tumors, therapeutic anti-CD40 antibodies would be of great value in treating disease.
- CD40 is a 50-kd type I transmembrane protein of the TNFR family that includes TNFR-1 , TNFR-2, CD30, CD27, 4-1BB, OX40, and Fas.
- the gene-encoding CD40 is located at chromosome 20 in humans and chromosome 2 in mice.
- CD40 is expressed widely on B cells, DCs, monocytes and macrophages, thymic epithelial cells, endothelial cells, mast cells, fibroblasts, and smooth muscle cells.
- CD40 ligand (CD154) is produced as a type Il transmembrane protein with an apparent molecular mass of 32 to 33 kd.
- TNF- ⁇ TNF- ⁇
- CD153 CD153
- CD70 CD70
- 4-1 BBL 4-1 BBL
- OX40L OX40L
- FasL Soluble forms of CD154 exist as 31 or 18-kd proteins, both of which retain the biologic activities of the membrane-bound CD154, and can potentially act as cytokines on distal CD40+ cells.
- the gene-encoding CD154 is located on the X chromosome in both humans and mice. Mutations in the CD154 gene result in the X-linked hyper-immunoglobulin (Ig) M syndrome in humans.
- CD154 expression originally thought to be restricted to activated T cells, mainly of the CD4 lineage, has now been identified on CD8+T cells, B cells, eosinophils, mast cells, basophils, DCs, and other cell types. Recently, platelets were found to express CD154 as well.
- CD154- CD40 pathway plays a key role in regulating thymus-dependent humoral responses. Indeed, patients with X-linked hyper-lgM syndrome have elevated IgM levels but low levels of IgA, IgG, and IgE; are devoid of the germinal centers; and are unable to mount thymus- dependent humoral responses. In mice, CD154-CD40 signaling was shown to be critical in regulating B-cell proliferation, Ig production, Ig class switching, rescue of B cells from apoptosis, germinal center formation, generation of B-cell memory, and clonal expansion and deletion of B cells.
- CD40 ligation by CD154 from T cells represents a key step in APC activation to enhance costimulatory molecule expression, which enables them to be competent to fully activate T cells.
- Induction of proinflammatory cytokines, including interleukin (IL) 12 by DCs, is another outcome of this APC interaction, which is critical for the development of TH 1 -type immune responses.
- IL interleukin
- CD154-CD40 interaction is bidirectional. Because in vitro CD154 ligation on T cells significantly enhances cytokine production, particularly of the TH2 type, cross-linking of CD154 on CD4 T cells in vivo contributes to the generation of helper function and germinal center. CD154-CD40 interaction provides the molecular basis for CD4 help in activation of cytotoxic CD8+ T cells, with DCs serving as a bridge between the 2 cell types. Although the molecular details of this 3-cell interaction have not been fully elucidated, it is now clear that activated CD4+ T cells stimulate DCs via CD154-CD40 and enable DCs to subsequently activate CD8+ T cells to differentiate into cytotoxic effectors.
- CD40 is expressed on many nonhematopoietic cells, particularly under the proinflammatory conditions.
- Cross-linking of CD40 on vascular endothelial cells activates these cells and increases expression of adhesion molecules, such as CD62E, CD106, CD54, and chemokines and cytokines, including IL-6 and IL-8. This may promote extravasation and accumulation of activated T cells at the sites of inflammation.
- platelets may provide another source of endothelial activation. Indeed, thrombin-activated platelets rapidly up-regulate CD154 expression, which may then interact with CD40 on endothelial cells and thus contribute to chemotactic effects from endothelial activation or damage, independently of T cells. This may have contributed, at least in part, to unexpected thromboembolic complications recently detected in primates and humans treated with some CD154 antibodies.
- CD154/CD40 signaling is also crucially important for controlling immunity and inflammation.
- the emerging picture indicates that ligation of the receptor CD40 via CD154, most potently in its trimeric form, functions in two ways.
- CD154 modulates physiologic processes, such as T cell-mediated effector functions and general immune responses required for appropriate host defense, but also triggers the expression of pro-inflammatory mediators, such as cytokines, adhesion molecules, and matrix degrading activities, all of which are associated with the pathogenesis of chronic inflammatory diseases, e.g., autoimmune disorders, arthritis, atherosclerosis, and cancer.
- CD40/CD154 interactions have advanced as a potential therapeutic target for these diseases, whereby two opposing strategies, interruption as well as enhancement of CD40 signaling, are explored for beneficial outcomes.
- CD40- CD40L interactions have thus been implicated in autoimmune diseases, such as atherosclerosis, asthma, systemic lupus erythematosus, multiple sclerosis, graft versus host disease, experimental autoimmune encephalitis and rheumatoid arthritis.
- Antibody efficacy may be due to a combination of these mechanisms, and their relative importance in clinical therapy for oncology appears to be cancer dependent.
- antibodies can mediate anti-tumor mechanism via growth inhibitory or apoptotic signaling that may occur when an antibody binds to its target on tumor cells. Such signaling may be potentiated when antibodies are presented to tumor cells bound to immune cells via Fc ⁇ R. Therefore increased affinity of antibodies to Fc ⁇ Rs may result in enhanced antiproliferative effects.
- ADCC antibody dependent cell-mediated cytotoxicity
- ADCP antibody dependent cell-mediated phagocytosis as used herein is meant the cell-mediated reaction wherein nonspecific cytotoxic cells that express Fc ⁇ Rs recognize bound antibody on a target cell and subsequently cause phagocytosis of the target cell.
- amino acid and “amino acid identity” as used herein is meant one of the 20 naturally occurring amino acids or any non-natural analogues that may be present at a specific, defined position.
- amino acid as used herein is both naturally occurring and synthetic amino acids.
- homophenylalanine, citrulline and noreleucine are considered amino acids for the purposes of the invention.
- amino acid also includes imino acid residues such as proline and hydroxyproline.
- the side chain may be in either the (R) or the (S) configuration. In a embodiment, the amino acids are in the (S) or L-configuration. If non-naturally occurring side chains are used, non-amino acid substituents may be used, for example to prevent or retard in vivo degradation.
- antibody herein is meant a protein consisting of one or more polypeptides substantially encoded by all or part of the recognized immunoglobulin genes.
- the recognized immunoglobulin genes include the kappa (K), lambda ( ⁇ ), and heavy chain genetic loci, which together comprise the myriad variable region genes, and the constant region genes mu ( ⁇ ), delta ( ⁇ ), gamma ( ⁇ ), sigma ( ⁇ ), and alpha ( ⁇ ) which encode the IgM, IgD, IgG (IgGI , lgG2, lgG3, and lgG4), IgE, and IgA (IgAI and lgA2) isotypes respectively.
- Antibody herein is meant to include full length antibodies and antibody fragments, and may refer to a natural antibody from any organism, an engineered antibody, or an antibody generated recombinantly for experimental, therapeutic, or other purposes.
- B cell or "B lymphocyte” as used herein is meant a type of lymphocyte developed in bone marrow that circulates in the blood and lymph, and provides humoral immunity. B cells recognize free antigen molecules and differentiate or mature into plasma cells that secrete immunoglobulin (antibodies) that inactivate the antigens. Memory cells are also generated that make the specific Immunoglobulin (antibody) on subsequent encounters with such antigen. B cells are also known as "Beta cells" in the islet of Langerhans.
- B-cell antigen or “B-cell marker” as used herein is meant any protein that is expressed on B cells.
- B-cell markers of the invention include CD40.
- CD40 as used herein is meant the protein encoded by the gene designated CD40.
- CD40 is also known as Tumor necrosis factor receptor superfamily member 5 (TNFRSF5), CD40L receptor, CD154 receptor, B-cell surface antigen CD40, CDw40, and Bp50.
- Human CD40 is designated GenelD:958 by Entrez Gene (Maglott et al., 2005, Nucleic Acids Res 33(Database Issue): D54-D58, and HGNC:11919 by HUGO (The Human Genome Organisation) Gene Nomenclature Committee (HGNC) (Wain et al., 2004, Genew: the Human Gene Nomenclature Database, 2004 updates, Nucleic Acids Res 32 Database issue:D255-7.
- the use of CD40 herein is meant to encompass all known or as yet undiscovered alleles and polymorphic forms of CD40.
- the sequence of human CD40 antigen used in the present study is provided in Figure 4, SEQ ID NO: 8.
- CDC complement dependent cytotoxicity
- constant region of an antibody as defined herein is meant the region of the antibody that is encoded by one of the light or heavy chain immunoglobulin constant region genes.
- constant light chain or “light chain constant region” as used herein is meant the region of an antibody encoded by the kappa (CK) or lambda (C ⁇ ) light chains.
- the constant light chain typically comprises a single domain, and as defined herein refers to positions 108-214 of CK or C ⁇ , wherein numbering is according to the EU index.
- constant heavy chain or “heavy chain constant region” as used herein is meant the region of an antibody encoded by the mu, delta, gamma, alpha, or epsilon genes to define the antibody's isotype as IgM, IgD, IgG, IgA, or IgE, respectively.
- the constant heavy chain as defined herein, refers to the N-terminus of the CH1 domain to the C-terminus of the CH3 domain, thus comprising positions 118-447, wherein numbering is according to the EU index.
- effector function as used herein is meant a biochemical event that results from the interaction of an antibody Fc region with an Fc receptor or ligand.
- Effector functions include Fc ⁇ R-mediated effector functions such as ADCC and ADCP, and complement- mediated effector functions such as CDC.
- effector cell as used herein is meant a cell of the immune system that expresses one or more Fc receptors and mediates one or more effector functions. Effector cells include but are not limited to monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans' cells, natural killer (NK) cells, and T cells, and may be from any organism including but not limited to humans, mice, rats, rabbits, and monkeys.
- Fab or "Fab region” as used herein is meant the polypeptides that comprise the V H , CH1 , V H , and C L immunoglobulin domains. Fab may refer to this region in isolation, or this region in the context of a full length antibody or antibody fragment.
- Fc or "Fc region”, as used herein is meant the polypeptide comprising the constant region of an antibody excluding the first constant region immunoglobulin domain. Thus Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains.
- Fc may include the J chain.
- Fc comprises immunoglobulin domains Cgamma2 and Cgamma3 (C ⁇ 2 and C ⁇ 3) and the hinge between Cgammai (C ⁇ 1) and Cgamma2 (C ⁇ 2).
- the human IgG heavy chain Fc region is usually defined to comprise residues C226 or P230 to its carboxyl-terminus, wherein the numbering is according to the EU index as in Kabat.
- Fc may refer to this region in isolation, or this region in the context of an Fc polypeptide, for example an antibody.
- Fc polypeptide as used herein is meant a polypeptide that comprises all or part of an Fc region.
- Fc polypeptides include antibodies, Fc fusions, isolated Fes, and Fc fragments.
- Fc gamma receptor or “Fc ⁇ R” as used herein is meant any member of the family of proteins that bind the IgG antibody Fc region and are substantially encoded by the Fc ⁇ R genes.
- this family includes but is not limited to Fc ⁇ RI (CD64), including isoforms Fc ⁇ RIa, Fc ⁇ RIb, and Fc ⁇ RIc; Fc ⁇ RII (CD32), including isoforms Fc ⁇ Rlla (including allotypes H131 and R131 ), Fc ⁇ Rllb (including Fc ⁇ Rllb-1 and Fc ⁇ Rllb-2), and Fc ⁇ Rllc; and Fc ⁇ RIII (CD16), including isoforms Fc ⁇ Rllla (including allotypes V158 and F158) and Fc ⁇ Rlllb (including allotypes Fc ⁇ Rlllb-NA1 and Fc ⁇ Rlllb-NA2) (Jefferis et ai, 2002, Immunol Lett 82:57-65, incorporated herein it its entirety by reference), as well as any undiscovered human Fc ⁇ Rs or Fc ⁇ R isoforms or allotypes.
- Mouse Fc ⁇ Rs include but are not limited to Fc ⁇ RI (CD64), Fc ⁇ RII (CD32), Fc ⁇ RIII (CD16), and Fc ⁇ RIII-2 (CD16-2), as well as any undiscovered mouse Fc ⁇ Rs or Fc ⁇ R isoforms or allotypes.
- An Fc ⁇ R may be from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys.
- Fc ligand or "Fc receptor” as used herein is meant a molecule, e.g., a polypeptide, from any organism that binds to the Fc region of an antibody to form an Fc- ligand complex.
- Fc ligands include but are not limited to Fc ⁇ Rs, FcRn, C1q, C3, mannan binding lectin, mannose receptor, staphylococcal protein A, streptococcal protein G, and viral Fc ⁇ R.
- Fc ligands also include Fc receptor homologs (FcRH), which are a family of Fc receptors that are homologous to the Fc ⁇ Rs (Davis et al., 2002, Immunological Reviews 190:123-136, incorporated herein it its entirety by reference).
- Fc ligands may include undiscovered molecules that bind Fc.
- IgG immunoglobulin gamma gene
- this class comprises IgGI , lgG2, lgG3, and lgG4.
- mice this class comprises IgGI , lgG2a, lgG2b, lgG3.
- immunoglobulin (Ig) herein is meant a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. Immunoglobulins include but are not limited to antibodies. Immunoglobulins may have a number of structural forms, including but not limited to full length antibodies, antibody fragments, and individual immunoglobulin domains.
- immunoglobulin domain herein is meant a region of an immunoglobulin that exists as a distinct structural entity as ascertained by one skilled in the art of protein structure. Ig domains typically have a characteristic ⁇ -sandwich folding topology. The known Ig domains in the IgG class of antibodies are V H , C ⁇ 1 , C ⁇ 2, C ⁇ 3, V L , and C L .
- modification herein is meant an alteration in the physical, chemical, or sequence properties of a protein, polypeptide, antibody, or immunoglobulin. Modifications of the invention are amino acid modifications and glycoform modifications.
- amino acid modification herein is meant an amino acid substitution, insertion, and/or deletion in a polypeptide sequence.
- amino acid substitution or “substitution” herein is meant the replacement of an amino acid at a particular position in a parent polypeptide sequence with another amino acid.
- substitution I332E refers to a variant polypeptide, in this case a constant heavy chain variant, in which the isoleucine at position 332 is replaced with glutamic acid.
- the WT residue may or may not be designated.
- 332E indicates the substitution of position 332 with a glutamic acid.
- multiple substitutions are typically separated by a slash.
- 239D/332E refers to a double variant comprising the substitutions 239D and 332E.
- amino acid insertion or “insertion” as used herein is meant the addition of an amino acid at a particular position in a parent polypeptide sequence.
- insert -236G designates an insertion of glycine at position 236.
- amino acid deletion or “deletion” as used herein is meant the removal of an amino acid at a particular position in a parent polypeptide sequence.
- G236- designates the deletion of glycine at position 236.
- glycoform modification or “modified glycoform” or “engineered qlycoform” as used herein is meant a carbohydrate composition that is covalently attached to a protein, for example an antibody, wherein the carbohydrate composition differs chemically from that of a parent protein.
- Modified glycoform typically refers to the different carbohydrate or oligosaccharide; thus for example an antibody may comprise a modified glycoform.
- modified glycoform may refer to the antibody that comprises the different carbohydrate or oligosaccharide.
- parent polypeptide a polypeptide that is subsequently modified to generate a variant, e.g., any polypeptide which serves as a template and/or basis for at least one amino acid modification described herein.
- the parent polypeptide may be a naturally occurring polypeptide, or a variant or engineered version of a naturally occurring polypeptide.
- Parent polypeptide may refer to the polypeptide itself, compositions that comprise the parent polypeptide, or the amino acid sequence that encodes it.
- parent antibody or “parent immunoglobulin” as used herein is meant an antibody or immunoglobulin that is modified to generate a variant (e.g., a parent antibody may include, but is not limited to, a protein comprising the constant region of a naturally occurring Ig).
- protein or "polypeptide” as used herein is meant at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides.
- the protein may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures, i.e. "analogs", such as peptoids.
- position as used herein is meant a location in the sequence of a protein.
- Positions may be numbered sequentially, or according to an established format, for example the EU index as in Kabat. Corresponding positions are determined as outlined herein, generally through alignment with other parent sequences.
- target antigen or “target” or “antigen” as used herein is meant the molecule that is bound specifically by the variable region of a given antibody.
- a target antigen may be a protein, carbohydrate, lipid, or other chemical compound.
- target cell as used herein is meant a cell that expresses a target antigen.
- variable region as used herein is meant the region of an immunoglobulin that comprises one or more Ig domains substantially encoded by any of the VK, V ⁇ , and/or V H genes that make up the kappa, lambda, and heavy chain immunoglobulin genetic loci respectively.
- variant protein protein variant
- variant polypeptide or “polypeptide variant” as used herein is meant a polypeptide sequence that differs from that of a parent polypeptide sequence by virtue of at least one amino acid modification or at least one glycoform modification.
- Variant polypeptide may refer to the polypeptide itself, a composition comprising the polypeptide, or the amino sequence that encodes it.
- the variant polypeptide has at least one amino acid modification compared to the parent polypeptide, e.g. from about one to about ten amino acid modifications, e.g., from about one to about five amino acid modifications compared to the parent.
- the variant polypeptide sequence herein may possess at least about 80% homology with a parent polypeptide sequence, e.g., at least about 90% homology, at least about 95% homology, etc.
- variable antibody or “antibody variant” as used herein is meant an antibody sequence that differs from that of a parent antibody sequence by virtue of at least one amino acid modification or at least one glycoform modification.
- Variant antibody or antibody variant may refer to the antibody polypeptide itself, compositions comprising the antibody variant polypeptide, or the amino acid sequence that encodes it.
- constant heavy chain variant or “constant light chain variant” or “Fc variant” as used herein is meant a constant heavy chain, constant light chain, or Fc region polypeptide or sequence, respectively, that differs in sequence from that of a parent sequence by virtue of at least one amino acid modification or at least one glycoform modification.
- wild type or WT herein is meant an amino acid sequence or a nucleotide sequence that is found in nature, including allelic variations.
- a WT protein, polypeptide, antibody, immunoglobulin, IgG, etc. has an amino acid sequence or a nucleotide sequence that has not been intentionally modified.
- EU index refers to the residue numbering of the human IgGI EU antibody, as described in Edelman et al., 1969, Biochemistry 63:78-85, incorporated herein it its entirety by reference.
- Antibodies are immunological proteins that bind a specific antigen. In most mammals, including humans and mice, antibodies are constructed from paired heavy and light polypeptide chains. The light and heavy chain variable regions show significant sequence diversity between antibodies, and are responsible for binding the target antigen. Each chain is made up of individual immunoglobulin (Ig) domains, and thus the generic term immunoglobulin is used for such proteins.
- Ig immunoglobulin
- Natural antibody structural units typically comprise a tetramer. Each tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one "light" chain (typically having a molecular weight of about 25 kDa) and one "heavy” chain (typically having a molecular weight of about 50-70 kDa).
- each of the light and heavy chains are made up of two distinct regions, referred to as the variable and constant regions.
- the heavy chain is composed of four immunoglobulin domains linked from N- to C-terminus in the order V H -CH1-CH2-CH3, referring to the heavy chain variable domain, heavy chain constant domain 1 , heavy chain constant domain 2, and heavy chain constant domain 3 respectively (also referred to as V H -C ⁇ 1-C ⁇ 2-C ⁇ 3, referring to the heavy chain variable domain, constant gamma 1 domain, constant gamma 2 domain, and constant gamma 3 domain respectively).
- the IgG light chain is composed of two immunoglobulin domains linked from N- to C-terminus in the order V L -C L , referring to the light chain variable domain and the light chain constant domain respectively.
- the constant regions show less sequence diversity, and are responsible for binding a number of natural proteins to elicit important biochemical events.
- variable region of an antibody contains the antigen binding determinants of the molecule, and thus determines the specificity of an antibody for its target antigen.
- the variable region is so named because it is the most distinct in sequence from other antibodies within the same class.
- three loops are gathered for each of the V domains of the heavy chain and light chain to form an antigen-binding site.
- Each of the loops is referred to as a complementarity-determining region (hereinafter referred to as a "CDR"), in which the variation in the amino acid sequence is most significant.
- CDR complementarity-determining region
- V H CDR1 variable region outside of the CDRs
- V H CDR2 variable region outside of the CDRs
- V L CDR3 variable region outside of the CDRs
- sequence variability does occur in the FR region between different antibodies.
- this characteristic architecture of antibodies provides a stable scaffold (the FR region) upon which substantial antigen binding diversity (the CDRs) can be explored by the immune system to obtain specificity for a broad array of antigens.
- a number of high-resolution structures are available for a variety of variable region fragments from different organisms, some unbound and some in complex with antigen.
- Antibodies are grouped into classes, also referred to as isotypes, as determined genetically by the constant region. Human constant light chains are classified as kappa (CK) and lambda (C ⁇ ) light chains.
- Human heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
- the IgG class is the most commonly used for therapeutic purposes.
- IgG as used herein is meant a polypeptide belonging to the class of antibodies that are substantially encoded by a recognized immunoglobulin gamma gene. In humans this class comprises subclasses IgGI , lgG2, lgG3, and lgG4. In mice this class comprises subclasses IgGI , lgG2a, lgG2b, lgG3.
- IgM has subclasses, including, but not limited to, IgMI and lgM2.
- IgA has several subclasses, including but not limited to IgAI and lgA2.
- isotype as used herein is meant any of the classes or subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions.
- the known human immunoglobulin isotypes are IgGI , lgG2, lgG3, lgG4, IgAI , lgA2, IgMI , lgM2, IgD, and IgE.
- Figure 1 provides the sequences of the human light chain kappa and heavy chain gamma constant chains.
- Figure 1 b & 1c shows an alignment of the human IgG constant heavy chains.
- IgGs that are hybrid compositions of the natural human IgG isotypes. Effector functions such as ADCC, ADCP, CDC, and serum half-life differ significantly between the different classes of antibodies, including for example human IgGI , lgG2, lgG3, lgG4, IgAI , lgA2, IgD, IgE, IgG, and IgM (Michaelsen et al., 1992, Molecular Immunology, 29(3): 319-326, entirely incorporated by reference).
- an lgG1/lgG3 hybrid variant may be constructed by substituting IgGI positions in the CH2 and CH3 region with the amino acids from lgG3 at positions where the two isotypes differ.
- hybrid variant IgG antibody may be constructed that comprises one or more substitutions selected from the group consisting of: 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N, 397M, 4221, 435R, and 436F, wherein numbering is according to the EU index.
- Such variant may provide alternate and/or improved effector function properties.
- relatively poor effector function of lgG2 may be improved by replacing key Fc ⁇ R binding residues with the corresponding amino acids in an IgG with better effector function.
- key residue differences between lgG2 and IgGI with respect to Fc ⁇ R binding may include P233, V234, A235, -236 (referring to a deletion in lgG2 relative to IgGI ), and G327.
- one or more amino acid modifications in the parent lgG2 wherein one or more of these residues is replaced with the corresponding IgGI amino acids, P233E, V234L, A235L, -236G (referring to an insertion of a glycine at position 236), and G327A, may provide enhanced effector function.
- the sequence of such an IgG, comprising a hybrid of residues from IgGI and lgG2, referred to herein as "Hybrid”, is provided in Figure 1.
- Gm polymorphism is determined by the IGHG1 , IGHG2 and IGHG3 genes which have alleles encoding allotypic antigenic determinants referred to as G1m, G2m, and G3m allotypes for markers of the human IgGI , lgG2 and lgG3 molecules (no Gm allotypes have been found on the gamma 4 chain). Markers may be classified into 'allotypes' and 'isoallotypes'. These are distinguished on different serological bases dependent upon the strong sequence homologies between isotypes.
- Allotypes are antigenic determinants specified by allelic forms of the Ig genes. Allotypes represent slight differences in the amino acid sequences of heavy or light chains of different individuals. Even a single amino acid difference can give rise to an allotypic determinant, although in many cases there are several amino acid substitutions that have occurred. Allotypes are sequence differences between alleles of a subclass whereby the antisera recognize only the allelic differences.
- An isoallotype is an allele in one isotype which produces an epitope which is shared with a non- polymorphic homologous region of one or more other isotypes and because of this the antisera will react with both the relevant allotypes and the relevant homologous isotypes (Clark, 1997, IgG effector mechanisms, Chem. Immunol. 65:88-110; Gorman & Clark, 1990, Semin. Immunol. 2(6):457-66, each incorporated herein it its entirety by reference). [104] Allelic forms of human immunoglobulins have been well-characterized (WHO Review of the notation for the allotypic and related markers of human immunoglobulins.
- G1 m (1 , 2, 3, 17) or G1m (a, x, f, z), G2m (23) or G2m (n), G3m (5, 6, 10, 11 , 13, 14, 15, 16, 21 , 24, 26, 27, 28) or G3m (b1 , c3, b5, b ⁇ , b3, b4, s, t, g1 , c5, u, v, g5)
- G1 m (1 , 2, 3, 17) or G1m (a, x, f, z), G2m (23) or G2m (n) or G3m (b1 , c3, b5, b ⁇ , b3, b4, s, t, g1 , c5, u, v, g5) (Lefranc, et al., The human IgG subclasses: molecular analysis of structure, function and regulation. Pergamon, Oxford, pp. 43-78 (1990); Lefranc, G. et
- FIG. 1 shows common haplotypes of the gamma chain of human IgGI ( Figure 2a) and lgG2 ( Figure 2b) showing the positions and the relevant amino acid substitutions.
- the antibodies of the present invention may be substantially encoded by any allotype, isoallotype, or haplotype of any immunoglobulin gene.
- Antibodies of the present invention may be substantially encoded by genes from any organism, e.g., mammals, including but not limited to humans, rodents including but not limited to mice and rats, lagomorpha including but not limited to rabbits and hares, camelidae including but not limited to camels, llamas, and dromedaries, and non-human primates, including but not limited to Prosimians, Platyrrhini (New World monkeys), Cercopithecoidea (Old World monkeys), and Hominoidea including the Gibbons and Lesser and Great Apes.
- the antibodies of the present invention are substantially human.
- the antibodies of the present invention may be substantially encoded by immunoglobulin genes belonging to any of the antibody classes.
- the antibodies of the present invention comprise sequences belonging to the IgG class of antibodies, including human subclasses IgGI, lgG2, lgG3, and lgG4.
- the antibodies of the present invention comprise sequences belonging to the IgA (including human subclasses IgAI and lgA2), IgD, IgE, IgG, or IgM classes of antibodies.
- the antibodies of the present invention may comprise more than one protein chain. That is, the present invention may find use in an antibody that is a monomer or an oligomer, including a homo- or hetero-oligomer.
- the antibodies of the invention are based on human IgG sequences, and thus human IgG sequences are used as the "base" sequences against which other sequences are compared, including but not limited to sequences from other organisms, for example rodent and primate sequences, as well as sequences from other immunoglobulin classes such as IgA, IgE, IgD, IgM, and the like. It is contemplated that, although the antibodies of the present invention are engineered in the context of one parent antibody, the variants may be engineered in or "transferred" to the context of another, second parent antibody.
- the amino acid sequence of a first antibody outlined herein is directly compared to the sequence of a second antibody. After aligning the sequences, using one or more of the homology alignment programs known in the art (for example using conserved residues as between species), allowing for necessary insertions and deletions in order to maintain alignment (i.e., avoiding the elimination of conserved residues through arbitrary deletion and insertion), the residues equivalent to particular amino acids in the primary sequence of the first antibody are defined. Alignment of conserved residues may conserve 100% of such residues.
- Equivalent residues may also be defined by determining structural homology between a first and second antibody that is at the level of tertiary structure for antibodies whose structures have been determined.
- equivalent residues are defined as those for which the atomic coordinates of two or more of the main chain atoms of a particular amino acid residue of the parent or precursor (N on N, CA on CA, C on C and O on O) are within 0.13 nm, e.g., 0.1 nm, after alignment. Alignment is achieved after the best model has been oriented and positioned to give the maximum overlap of atomic coordinates of non-hydrogen protein atoms of the proteins.
- the antibodies discovered by the present invention may be engineered into any second parent antibody that has significant sequence or structural homology with the antibody.
- the variant antibody may be engineered in a human lgG2 parent antibody, a human IgA parent antibody, a mouse lgG2a or lgG2b parent antibody, and the like.
- the context of the parent antibody does not affect the ability to transfer the antibodies of the present invention to other parent antibodies.
- the variant antibodies that are engineered in a human IgGI antibody that targets one antigen epitope may be transferred into a human lgG2 antibody that targets a different antigen epitope, and so forth.
- immunoglobulin domains there are several immunoglobulin domains in the heavy chain.
- immunoglobulin (Ig) domain herein is meant a region of an immunoglobulin having a distinct tertiary structure.
- the domains of the constant heavy chain including, the constant heavy (CH) domains and the hinge.
- the IgG isotypes each have three CH regions: “CH1” refers to positions 118-220, “CH2” refers to positions 237-340, and “CH3" refers to positions 341-447 according to the EU index as in Kabat.
- hinge or “hinge region” or “antibody hinge region” or “immunoglobulin hinge region” herein is meant the flexible polypeptide comprising the amino acids between the first and second constant domains of an antibody. Structurally, the IgG CH 1 domain ends at EU position 220, and the IgG CH2 domain begins at residue EU position 237.
- the hinge is herein defined to include positions 221 (D221 in IgGI ) to 236 (G236 in IgGI ), wherein the numbering is according to the EU index as in Kabat.
- the lower hinge is included, with the “lower hinge” generally referring to positions 226 or 230.
- the constant heavy chain refers to the N-terminus of the CH1 domain to the C-terminus of the CH3 domain, thus comprising positions 118-447, wherein numbering is according to the EU index.
- the constant light chain comprises a single domain, and as defined herein refers to positions 108-214 of CK or C ⁇ , wherein numbering is according to the EU index.
- full-length antibodies are full-length antibodies.
- full length antibody herein is meant the structure that constitutes the natural biological form of an antibody, including variable and constant regions.
- the full length antibody of the IgG class is a tetramer and consists of two identical pairs of two immunoglobulin chains, each pair having one light and one heavy chain, each light chain comprising immunoglobulin domains V L and C L , and each heavy chain comprising immunoglobulin domains V H , CH1 (C ⁇ 1), CH2 (C ⁇ 2), and CH3 (C ⁇ 3).
- IgG antibodies may consist of only two heavy chains, each heavy chain comprising a variable domain attached to the Fc region.
- the antibodies can be a variety of structures, including, but not limited to antibody fragments.
- Antibody fragments include but are not limited to bispecific antibodies, minibodies, domain antibodies, synthetic antibodies, antibody mimetics, chimeric antibodies, antibody fusions (sometimes referred to as "antibody conjugates"), and fragments of each, respectively.
- Specific antibody fragments include, but are not limited to, (i) the Fab fragment consisting of VL, VH, CL and CH1 domains, (ii) the Fd fragment consisting of the VH and CH1 domains, (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment, which consists of a single variable region, (v) isolated CDR regions, (vi) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (viii) bispecific single chain Fv dimers and (ix) "diabodies” or "triabodies", multivalent or multispecific fragments constructed by gene fusion.
- the antibody fragments may be modified.
- the molecules may be stabilized by the incorporation of disulfide bridges linking the VH and VL domains.
- Examples of antibody formats and architectures are described in Holliger & Hudson, 2006, Nature Biotechnology 23(9): 1126- 1136, and Carter 2006, Nature Reviews Immunology 6:343-357 and references cited therein, all expressly incorporated by reference.
- Antibodies of the invention may include multispecific antibodies, notably bispecific antibodies, also sometimes referred to as "diabodies". These are antibodies that bind to two (or more) different antigens. Diabodies can be manufactured in a variety of ways known in the art, e.g., prepared chemically or from hybrid hybridomas.
- the antibody is a minibody.
- Minibodies are minimized antibody-like proteins comprising a scFv joined to a CH3 domain.
- the scFv can be joined to the Fc region, and may include some or all of the hinge region.
- the antibody of the invention is an antibody fragment.
- antibodies that comprise Fc regions, Fc fusions, and the constant region of the heavy chain (CH1-hinge-CH2-CH3).
- Antibodies of the present invention may comprise Fc fragments.
- An Fc fragment of the present invention may comprise from 1 - 90% of the Fc region, e.g., 10 - 90%, 30 - 90%, etc.
- an Fc fragment of the present invention may comprise an IgGI C ⁇ 2 domain, an IgGI C ⁇ 2 domain and hinge region, an IgGI C ⁇ 3 domain, and so forth.
- an Fc fragment of the present invention additionally comprises a fusion partner, effectively making it an Fc fragment fusion.
- Fc fragments may or may not contain extra polypeptide sequence.
- lmmunogenicity is the result of a complex series of responses to a substance that is perceived as foreign, and may include production of neutralizing and non-neutralizing antibodies, formation of immune complexes, complement activation, mast cell activation, inflammation, hypersensitivity responses, and anaphylaxis.
- protein immunogenicity Several factors can contribute to protein immunogenicity, including but not limited to protein sequence, route and frequency of administration, and patient population. Immunogenicity may limit the efficacy and safety of a protein therapeutic in multiple ways. Efficacy can be reduced directly by the formation of neutralizing antibodies. Efficacy may also be reduced indirectly, as binding to either neutralizing or non-neutralizing antibodies typically leads to rapid clearance from serum. Severe side effects and even death may occur when an immune reaction is raised.
- the scaffold components can be a mixture from different species.
- Such antibody may be a chimeric antibody and/or a humanized antibody.
- both “chimeric antibodies” and “humanized antibodies” refer to antibodies that combine regions from more than one species.
- “Chimeric antibodies” traditionally comprise variable region(s) from a mouse (or rat, in some cases) and the constant region(s) from a human (Morrison et al., 1984, Proc Natl Acad Sci USA 81 : 6851-6855, incorporated herein it its entirety by reference).
- humanized antibody as used herein is meant an antibody comprising a human framework region (FR) and one or more complementarity determining regions (CDRs) from a non-human (usually mouse or rat) antibody.
- the non-human antibody providing the CDRs is called the “donor” and the human immunoglobulin providing the framework is called the “acceptor”.
- Humanization relies principally on the grafting of donor CDRs onto acceptor (human) VL and VH frameworks (Winter US 5225539, incorporated herein it its entirety by reference). This strategy is referred to as "CDR grafting".
- the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin, and thus will typically comprise a human Fc region.
- An immunoglobulin constant region typically that of a human immunoglobulin
- a variety of techniques and methods for humanizing and reshaping non-human antibodies are well known in the art (See Tsurushita & Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biology of B Cells, 533-545, Elsevier Science (USA), and references cited therein, each incorporated herein it its entirety by reference).
- Humanization or other methods of reducing the immunogenicity of nonhuman antibody variable regions may include resurfacing methods, as described for example in Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91 :969-973, incorporated herein it its entirety by reference.
- selection based methods may be employed to humanize and/or affinity mature antibody variable regions, that is, to increase the affinity of the variable region for its target antigen.
- Other humanization methods may involve the grafting of only parts of the CDRs, including but not limited to methods described in USSN 09/810,502; Tan et al., 2002, J. Immunol. 169:1119-1125; De Pascalis et al., 2002, J. Immunol. 169:3076-3084, incorporated herein it its entirety by reference.
- Structure-based methods may be employed for humanization and affinity maturation, for example as described in USSN 10/153,159 and related applications, each incorporated herein it its entirety by reference.
- the immunogenicity of the antibody is reduced using a method described in USSN 11/004,590, entitled “Methods of Generating Variant Proteins with Increased Host String Content and Compositions Thereof, filed on December 3, 2004, incorporated herein it its entirety by reference.
- Modifications to reduce immunogenicity may include modifications that reduce binding of processed peptides derived from the parent sequence to MHC proteins. For example, amino acid modifications would be engineered such that there are no or a minimal number of immune epitopes that are predicted to bind, with high affinity, to any prevalent MHC alleles.
- the antibodies of the present invention may be fully human, that is the sequences of the antibodies are completely or substantially human.
- Fully human antibody or “complete human antibody” refers to a human antibody having the gene sequence of an antibody derived from a human chromosome with the modifications outlined herein.
- the antibodies of the present invention may be virtually any antibody that binds to CD40, e.g., may comprise the variable regions (e.g., the CDRs) of any known or undiscovered anti-CD40 antibody.
- Antibodies of the invention may display selectivity for CD40.
- Examples include full-length versus splice variants, cell-surface vs. soluble forms, selectivity for various polymorphic variants, or selectivity for specific conformational forms of a target.
- An antibody of the present invention may bind any epitope or region on CD40 and may be specific for fragments, mutant forms, splice forms, or aberrant forms of the antigens.
- a number of useful antibodies have been discovered that target CD40 that may find use in the present invention.
- Suitable CD40 antibodies or immunoadhesins include the CD40 antibodies or immunoadhesins S2C6 (Paulie et al., Cancer Immunol. Immunother., Vo1 17, No 3, (1984) pp.
- the antibody of the invention is a therapeutic, a diagnostic, or a research reagent. In one embodiment, an antibody of the invention is a therapeutic. Alternatively, the antibody of the present invention may be used for agricultural or industrial uses. An antibody of the present invention may find use in an antibody composition that is monoclonal or polyclonal.
- the antibodies of the present invention may be agonists, antagonists, neutralizing, inhibitory, or stimulatory. In one embodiment, the antibodies of the present invention are used to kill target cells that bear the target antigen, for example cancer cells. In an alternate embodiment, the antibodies of the present invention are used to block, antagonize, or agonize the target antigen.
- the antibodies of the present invention are used to block, antagonize, or agonize the target antigen and kill the target cells that bear the target antigen.
- Modifications for optimizing effector function [124] The present invention is directed to antibodies comprising modifications, wherein the modifications alter affinity to one or more Fc receptors, and/or alter the ability of the antibody to mediate one or more effector functions. Modifications of the invention include amino acid modifications and glycoform modifications. The Fc portion disclosed herein can be combined with any anti-CD40 disclosed herein. [125] Amino acid modifications
- variants that alter binding to one or more human Fc receptors may comprise an amino acid modification in the heavy chain constant region, as described herein, selected from the group consisting of 221K, 221Y, 222E, 222Y, 223E, 223K, 224E, 224Y, 225E, 225K, 225W, 227E, 227G, 227K, 227Y, 228E, 228G, 228K, 228Y, 230A, 230E, 230G, 230Y, 231 E, 231 G, 231 K, 231 P, 231 Y, 232E, 232G, 232K, 232Y, 233A, 233D, 233F, 233G, 233H, 233I, 233K, 233L, 233M, 233N, 233Q, 233R, 233S, 233T, 233V, 233W, 233Y, 234A, 234D, 234E, 2
- variants that alter binding to one or more human Fc receptors may comprise an amino acid modification in the heavy chain constant region, as described herein, selected from the group consisting of 118K, 118E, 118Y, 119R, 119E, 119Y 1 120R, 120E, 1201, 121E, 121Y, 121 H, 122E, 122R, 124K, 124E, 124Y, 126K, 126D, 129L, 129D, 131G, 131T, 132D, 132R, 132L, 133R, 133E, 133L, 1351, 135E, 135K, 136E, 136K, 1361, 137E, 138S, 138R, 138D, 1391, 139E 1 139K, 147A, 147E 1 148Y, 148K, 150L 1 150K, 150E 1 151 A, 151 D 1 152L 1 152K, 153L, 153D,
- variants that alter binding to one or more human Fc receptors may comprise an amino acid modification in the light chain constant region, as described herein, selected from the group consisting of 108D, 1081, 108Q, 109D, 109P, 109R, 110E, 1101, 110K, 111 E, 111 K, 111 L, 112E, 112R, 112Y, 114D, 1141, 114K, 116T, 121 D, 122R, 122S, 122Y, 123L, 123R, 124E, 125E, 125K, 126D, 126L, 126Q, 127A, 127D, 127K, 128N, 129E, 1291, 129K, 131T, 137K, 137S, 138D, 138K, 138L, 140E, 140H, 140K, 141E 1 141 K, 142D, 142G, 142L, 143A, 143L, 143R,
- Additional substitutions that may also be used in the present invention include other substitutions that modulate Fc receptor affinity, Fc ⁇ R-mediated effector function, and/or complement mediated effector function include but are not limited to 298A, 298T, 326A, 326D, 326E 1 326W, 326Y, 333A, 333S 1 334L, and 334A (US 6,737,056; Shields et al, Journal of Biological Chemistry, 2001 , 276(9):6591-6604; US 6,528,624; ldusogie et al., 2001 , J.
- antibodies of the present invention may be combined with constant heavy chain variants that alter FcRn binding. These include modifications that modify FcRn affinity in a pH-specific manner.
- variants that increase Fc binding to FcRn include but are not limited to: 250E, 250Q, 428L, 428F, 250Q/428L (Hinton et al., 2004, J. Biol. Chem. 279(8): 6213-6216, Hinton et al.
- antibodies may comprise isotypic modifications, that is modifications in a parent IgG to the amino acid type in an alternate IgG.
- an lgG1/lgG3 hybrid variant may be constructed by substituting IgGI positions in the CH2 and/or CH3 region with the amino acids from lgG3 at positions where the two isotypes differ.
- a hybrid variant IgG antibody may be constructed that comprises one or more substitutions selected from the group consisting of: 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N, 397M, 4221, 435R, and 436F.
- an lgG1/lgG2 hybrid variant may be constructed by substituting lgG2 positions in the CH2 and/or CH3 region with amino acids from IgGI at positions where the two isotypes differ.
- a hybrid variant IgG antibody may be constructed that comprises one or more modifications selected from the group consisting of 233E, 234L, 235L, -236G (referring to an insertion of a glycine at position 236), and 327A.
- the present invention is directed to antibodies comprising means for altering affinity to one or more Fc receptors, and/or alter the ability of the antibody to mediate one or more effector functions.
- Means of the invention include amino acid modifications (e.g., positional means for optimizing effector function, substitutional means for optimizing effector function, etc.) and glycoform modifications (e.g., means for glycoform modifications).
- Amino acid modifications e.g., positional means for optimizing effector function, substitutional means for optimizing effector function, etc.
- glycoform modifications e.g., means for glycoform modifications.
- positional means for optimizing effector function include but is not limited to, modification of an amino acid at one or more heavy chain constant region positions (e.g., at positions 221 , 222, 223, 224, 225, 227, 228, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241 , 243, 244, 245, 246, 247, 249, 255, 258, 260, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271 , 272, 273, 274, 275, 276, 278, 280, 281 , 282, 283, 284, 285, 286, 288, 290, 291 , 292, 293, 294, 295, 296, 297, 298, 299, 300, 301 , 302, 303, 304, 305
- substitutional means for optimizing effector functions, for altering binding to one or more human Fc receptors include, but is not limited to, a substitution of an amino acid at one or more heavy chain constant region positions, e.g., one or more of the following amino acid substitutions in the heavy chain constant region positions: 221 K, 221 Y, 222E, 222Y, 223E, 223K, 224E, 224Y, 225E, 225K, 225W, 227E, 227G, 227K, 227Y, 228E, 228G, 228K 1 228Y, 230A, 230E, 230G, 230Y, 231 E, 231 G, 231 K, 231 P, 231 Y, 232E, 232G, 232K, 232Y, 233A, 233D, 233F, 233G, 233H, 233I, 233K, 233L, 233M, 233N, 233Q, 233
- positional means for optimizing effector function include, but is not limited to, modification of an amino acid at one or more heavy chain constant region positions (e.g., at positions 118, 119, 120, 121 , 122, 124, 126, 129, 131 , 132, 133, 135, 136, 137, 138, 139, 147, 148, 150, 151 , 152, 153, 155, 157, 159, 160, 161 , 162, 163, 164, 165, 166, 167, 168, 169, 171 , 172, 173, 174, 175, 176, 177, 178, 179, 180, 183, 187, 188, 189, 190, 191 , 192, 193, 194, 195, 196, 197, 198
- positional means for optimizing effector function include but is not limited to, modification of an amino acid at one or more light chain constant region positions (e.g., at positions 108, 109, 110, 111 , 112, 114, 116, 121 , 122, 123, 124, 125, 126, 127, 128, 129, 131, 137, 138, 140, 141 , 142, 143, 145, 147, 149, 150, 151 , 152, 153, 154, 155, 156, 157, 159, 160, 161 , 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 176, 180, 181 , 182, 183, 184, 185,
- substitional means for altering binding to one or more human Fc receptors include, but is not limited to, a substition of an amino acid at one or more heavy chain constant region positions, e.g., one or more of the following substitutions: 118K, 118E, 118Y, 119R, 119E, 119Y, 120R, 120E, 1201, 121 E, 121 Y, 121 H, 122E, 122R, 124K, 124E, 124Y, 126K, 126D, 129L, 129D, 131G, 131T, 132D, 132R, 132L, 133R, 133E, 133L, 1351, 135E, 135K, 136E, 136K, 1361, 137E, 138S, 138R, 138D, 1391, 139E, 139K, 147A, 147E, 148Y, 148K, 150L, 150K, 150E,
- substitutional means for altering binding to one or more human Fc receptors include, but is not limited to, a substition of an amino acid modification at one or more light chain constant region positions, e.g., one or more of the following amino acid substitutions in the light chain constant region positions: 108D, 1081, 108Q, 109D, 109P, 109R 1 110E, 1101, 110K, 111 E, 111 K 1 111 L 1 112E 1 112R, 112Y, 114D, 1141, 114K, 116T 1 121 D 1 122R 1 122S, 122Y, 123L, 123R, 124E, 125E, 125K 1 126D 1 126L 1 126Q, 127A, 127D 1 127K 1 128N 1 129E, 1291, 129K, 131T, 137K, 137S, 138D, 138K, 138L, 140E, 140H, 140K, 141
- Additional substitutional means that may also be used in the present invention include substitutional means for modulating Fc receptor affinity, Fc ⁇ R-mediated effector function, and/or complement mediated effector function, e.g., one or more of the following amino acid substitutions: 298A, 298T, 326A, 326D, 326E, 326W, 326Y, 333A, 333S, 334L, and 334A (US 6,737,056; Shields et al, Journal of Biological Chemistry, 2001 , 276(9):6591- 6604; US 6,528,624; ldusogie et al., 2001 , J.
- substitutional means for modulating Fc receptor affinity, Fc ⁇ R-mediated effector function, and/or complement mediated effector function e.g., one or more of the following amino acid substitutions: 298A, 298T, 326A, 326D, 326E, 326W, 326Y, 333A, 333S, 334L
- antibodies of the present invention may be combined with means for altering FcRn binding, e.g., antibodies of the present invention may be combined with constant heavy chain variants . These include means for modifying FcRn affinity in a pH-specific manner.
- substitutional means for increasing Fc binding to FcRn include, but are not limited to, one or more of the following amino acid substitutions: 250E, 250Q, 428L, 428F, 250Q/428L (Hinton et al., 2004, J. Biol. Chem. 279(8): 6213-6216, Hinton et al.
- antibodies may comprise means for isotypic modifications, that is modifications in a parent IgG to the amino acid type in an alternate IgG.
- an lgG1/lgG3 hybrid variant may be constructed by a substitutional means for substituting IgGI positions in the CH2 and/or CH3 region with the amino acids from lgG3 at positions where the two isotypes differ.
- a hybrid variant IgG antibody may be constructed that comprises one or more substitutional means, e.g., 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N, 397M, 4221, 435R, and 436F.
- an lgG1/lgG2 hybrid variant may be constructed by a substitutional means for substituting lgG2 positions in the CH2 and/or CH3 region with amino acids from IgGI at positions where the two isotypes differ.
- a hybrid variant IgG antibody may be constructed that comprises one or more substitutional means, e.g., one or more of the following amino acid substations: 233E, 234L, 235L, -236G (referring to an insertion of a glycine at position 236), and 327A.
- polypeptides including antibodies, are subjected to a variety of post- translational modifications involving carbohydrate moieties, such as glycosylation with oligosaccharides. There are several factors that can influence glycosylation. The species, tissue and cell type have all been shown to be important in the way that glycosylation occurs. In addition, the extracellular environment, through altered culture conditions such as serum concentration, may have a direct effect on glycosylation. (Lifely et a/., 1995, Glycobiology 5(8): 813-822), incorporated herein it its entirety by reference. [149] All antibodies contain carbohydrate at conserved positions in the constant regions of the heavy chain. Each antibody isotype has a distinct variety of N-linked carbohydrate structures.
- IgG Aside from the carbohydrate attached to the heavy chain, up to 30% of human IgGs have a glycosylated Fab region. IgG has a single N-linked biantennary carbohydrate at Asn297 of the CH2 domain. For IgG from either serum or produced ex vivo in hybridomas or engineered cells, the IgG are heterogeneous with respect to the Asn297 linked carbohydrate (Jefferis et a/., 1998, Immunol. Rev. 163:59-76; Wright et a/., 1997, Trends Biotech 15:26-32, each incorporated herein it its entirety by reference).
- the core oligosaccharide normally consists of GlcNAc 2 Man 3 GlcNAc, with differing numbers of outer residues.
- the carbohydrate moieties of the present invention will be described with reference to commonly used nomenclature for the description of oligosaccharides. A review of carbohydrate chemistry which uses this nomenclature is found in Hubbard et al. 1981 , Ann. Rev. Biochem. 50:555-583, incorporated herein it its entirety by reference.
- This nomenclature includes, for instance, Man, which represents mannose; GIcNAc, which represents 2-N-acetylglucosamine; GaI which represents galactose; Fuc for fucose; and GIc, which represents glucose.
- Sialic acids are described by the shorthand notation NeuNAc, for 5-N-acetylneuraminic acid, and NeuNGc for 5-glycolylneuraminic.
- the term "glycosylation” is the attachment of oligosaccharides (carbohydrates containing two or more simple sugars linked together e.g. from two to about twelve simple sugars linked together) to a glycoprotein.
- the oligosaccharide side chains are typically linked to the backbone of the glycoprotein through either N- or O-linkages.
- the oligosaccharides of the present invention occur generally are attached to a CH2 domain of an Fc region as N- linked oligosaccharides.
- N-linked glycosylation refers to the attachment of the carbohydrate moiety to an asparagine residue in a glycoprotein chain.
- each of murine IgGI , lgG2a, lgG2b and lgG3 as well as human IgGI , lgG2, lgG3, lgG4, IgA and IgD CH2 domains have a single site for N-linked glycosylation at amino acid residue 297 (Kabat et al. Sequences of Proteins of Immunological Interest, 1991, incorporated herein it its entirety by reference).
- a "mature core carbohydrate structure” refers to a processed core carbohydrate structure attached to an Fc region which generally consists of the following carbohydrate structure GlcNAc(Fucose)-GlcNAc-Man-(Man-GlcNAc) 2 typical of biantennary oligosaccharides.
- the mature core carbohydrate structure is attached to the Fc region of the glycoprotein, generally via N-linkage to Asn297 of a CH2 domain of the Fc region.
- a "bisecting GIcNAc” is a GIcNAc residue attached to the ⁇ 1 ,4 mannose of the mature core carbohydrate structure.
- the bisecting GIcNAc can be enzymatically attached to the mature core carbohydrate structure by a ⁇ (1 ,4)-N-acetylglucosaminyltransferase III enzyme (GnTIII).
- GnTIII ⁇ (1 ,4)-N-acetylglucosaminyltransferase III enzyme
- CHO cells do not normally express GnTIII (Stanley et al., 1984, J. Biol. Chem. 261 :13370-13378), but may be engineered to do so (Umana et al., 1999, Nature Biotech. 17:176-180).
- modified glycoforms or engineered glycoforms
- modified glycoform or engineered glycoform as used herein is meant a carbohydrate composition that is covalently attached to a protein, for example an antibody, wherein the carbohydrate composition differs chemically from that of a parent protein.
- Engineered glycoforms may be useful for a variety of purposes, including but not limited to enhancing or reducing Fc ⁇ R-mediated effector function.
- the antibodies of the present invention are modified to control the level of fucosylated and/or bisecting oligosaccharides that are covalently attached to the Fc region.
- the FUT8 gene encodes the ⁇ 1 ,6-fucosyltransferase enzyme that catalyzes the transfer of a fucosyl residue from GDP-fucose to position 6 of Asn-linked (N-Iinked) GlcNac of an N-glycan (Yanagidani et al., 1997, J Biochem 121 :626-632). It is known that the ⁇ 1 ,6- fucosyltransferase is the only enzyme responsible for adding fucose to the N-linked biantennary carbohydrate at Asn297 in the CH2 domain of the IgG antibody.
- These techniques control the level of fucosylated and/or bisecting oligosaccharides that are covalently attached to the Fc region, for example by expressing an IgG in various organisms or cell lines, engineered or otherwise (for example Lec-13 CHO cells or rat hybridoma YB2/0 cells), by regulating enzymes involved in the glycosylation pathway (for example FUT8 [ ⁇ 1 ,6-fucosyltranserase] and/or ⁇ 1-4- N- acetylglucosaminyltransferase III [GnTIII]), or by modifying carbohydrate(s) after the IgG has been expressed.
- enzymes involved in the glycosylation pathway for example FUT8 [ ⁇ 1 ,6-fucosyltranserase] and/or ⁇ 1-4- N- acetylglucosaminyltransferase III [GnTIII]
- Methods for modifying glycoforms include but are not limited to using a glycoengineered strain of yeast Pichia pastoris (Li et al., 2006, Nature Biotechnology 24(2):210-215), a glycoengineered strain of the moss Physcomitrella patens wherein the enzymes ⁇ 1 ,2-xylosyltransferase and/or ⁇ 1 ,3-fucosyltransferase are knocked out in (Nechansky et al., 2007, MoI. Immunol.
- Engineered glycoform typically refers to the different carbohydrate or oligosaccharide; thus for example an antibody may comprise an engineered glycoform. Alternatively, engineered glycoform may refer to the antibody that comprises the different carbohydrate or oligosaccharide.
- a "parent antibody” is a glycosylated antibody having the same amino acid sequence and mature core carbohydrate structure as an engineered glycoform of the present invention, except that fucose is attached to the mature core carbohydrate structure of the parent antibody.
- a composition comprising the parent glycoprotein about 50-100% or about 70-100% of the parent glycoprotein comprises a mature core carbohydrate structure having fucose attached thereto.
- the present invention provides a composition comprising a glycosylated antibody having an Fc region, wherein about 51-100% of the glycosylated antibody in the composition comprises a mature core carbohydrate structure which lacks fucose, attached to the Fc region of the antibody.
- about 80-100% of the antibody in the composition comprises a mature core carbohydrate structure which lacks fucose.
- about 90-99% of the antibody in the composition lacks fucose attached to the mature core carbohydrate structure.
- the antibody in the composition both comprises a mature core carbohydrate structure that lacks fucose and additionally comprises at least one amino acid modification in the Fc region.
- the combination of engineered glycoform and amino acid modification provides optimal Fc receptor binding properties to the anti-CD40 antibody.
- the present invention provides variant antibodies that are optimized for a number of therapeutically relevant properties.
- a variant antibody comprises one or more amino acid modifications or gly ⁇ oform modifications relative to a parent antibody, wherein the amino acid modification(s) provide one or more optimized properties.
- the antibodies of the present invention are variant antibodies.
- An antibody of the present invention differs in amino acid sequence from its parent antibody by virtue of at least one amino acid or glycoform modification.
- variant antibodies of the present invention have at least one amino acid or glycoform modification compared to the parent.
- the variant antibodies of the present invention may have more than one amino acid modification as compared to the parent, for example from about one to fifty amino acid modifications, e.g., from about one to ten amino acid modifications, from about one to about five amino acid modifications, etc., compared to the parent.
- the sequences of the variant antibodies and those of the parent antibodies are substantially homologous.
- the variant antibody sequences herein will possess about 80% homology with the parent antibody sequence, e.g., at least about 90% homology, e at least about 95% homology, etc.
- the antibodies of the present invention comprise amino acid modifications that provide optimized effector function properties relative to the parent. Substitutions and optimized effector function properties are described in USSN 10/672,280, PCT US03/30249, and USSN 10/822,231 , and USSN 60/627,774, filed 11/12/2004 and entitled Optimized Fc Variants". Properties that may be optimized include but are not limited to enhanced or reduced affinity for an Fc ⁇ R. In one embodiment, the antibodies of the present invention are optimized to possess enhanced affinity for a human activating Fc ⁇ R, e.g., FcyRI, Fc ⁇ Rlla, Fc ⁇ Rllc, Fc ⁇ Rllla, and Fc ⁇ Rlllb.
- an antibody of the invention are optimized to possess enhanced affinity for a human Fc ⁇ Rllla.
- the antibodies are optimized to possess reduced affinity for the human inhibitory receptor Fc ⁇ Rllb. These embodiments are anticipated to provide antibodies with enhanced therapeutic properties in humans, for example enhanced effector function and greater anti-cancer potency.
- the antibodies of the present invention are optimized to have reduced or ablated affinity for a human Fc ⁇ R, including but not limited to Fc ⁇ RI, Fc ⁇ Rlla, Fc ⁇ Rllb, Fc ⁇ Rllc, Fc ⁇ Rllla, and Fc ⁇ Rlllb. These embodiments are anticipated to provide antibodies with enhanced therapeutic properties in humans, for example reduced effector function and reduced toxicity.
- antibodies of the present invention provide enhanced affinity for one or more Fc ⁇ Rs, yet reduced affinity for one or more other Fc ⁇ Rs.
- an antibody of the present invention may have enhanced binding to Fc ⁇ Rllla, yet reduced binding to Fc ⁇ Rllb.
- an antibody of the present invention may have enhanced binding to Fc ⁇ Rlla and Fc ⁇ RI, yet reduced binding to Fc ⁇ Rllb.
- an antibody of the present invention may have enhanced affinity for Fc ⁇ Rllb, yet reduced affinity to one or more activating Fc ⁇ Rs.
- the modifications of the invention may enhance binding affinity for one or more Fc ⁇ Rs.
- an Fc variant binds to an Fc receptor with a significantly higher equilibrium constant of association (K 3 ) or lower equilibrium constant of dissociation (K d ) than the parent polypeptide when the amounts of variant and parent polypeptide in the binding assay are essentially the same.
- the Fc variant with improved Fc ⁇ R binding affinity may display from about 5 fold to about 1000 fold, e.g. from about 10 fold to about 500 fold improvement in Fc receptor binding affinity compared to the parent polypeptide, where Fc receptor binding affinity is determined, for example, as disclosed in the Examples herein.
- Embodiments comprise optimization of Fc binding to a human Fc ⁇ R, however in alternate embodiments the antibodies of the present invention possess enhanced or reduced affinity for Fc ⁇ Rs from nonhuman organisms, including but not limited to rodents and non- human primates. Antibodies that are optimized for binding to a nonhuman Fc ⁇ R may find use in experimentation. For example, mouse models are available for a variety of diseases that enable testing of properties such as efficacy, toxicity, and pharmacokinetics for a given drug candidate.
- cancer cells can be grafted or injected into mice to mimic a human cancer, a process referred to as xenografting.
- Testing of antibodies that comprise antibodies that are optimized for one or more mouse Fc ⁇ Rs may provide valuable information with regard to the efficacy of the protein, its mechanism of action, and the like.
- the antibodies of the present invention may also be optimized for enhanced functionality and/or solution properties in aglycosylated form.
- the aglycosylated antibodies of the present invention bind an Fc ligand with greater affinity than the aglycosylated form of the parent antibody.
- the Fc ligands include but are not limited to Fc ⁇ Rs, C1q, FcRn, and proteins A and G, and may be from any source including but not limited to human, mouse, rat, rabbit, or monkey.
- the antibodies are optimized to be more stable and/or more soluble than the aglycosylated form of the parent antibody.
- Antibodies of the invention may comprise modifications that modulate interaction with Fc ligands other than Fc ⁇ Rs, including but not limited to complement proteins, FcRn, and Fc receptor homologs (FcRHs).
- FcRHs include but are not limited to FcRHI , FcRH2, FcRH3, FcRH4, FcRH5, and FcRH ⁇ (Davis et ai, 2002, Immunol. Reviews 190:123-136, incorporated herein it its entirety by reference).
- the Fc ligand specificity of the antibody of the present invention will determine its therapeutic utility.
- the utility of a given antibody for therapeutic purposes will depend on the epitope or form of the target antigen and the disease or indication being treated.
- enhanced Fc ⁇ R-mediated effector functions may be desirable. This may be particularly favorable for anti-cancer antibodies.
- antibodies may be used that comprise antibodies that provide enhanced affinity for activating Fc ⁇ Rs and/or reduced affinity for inhibitory Fc ⁇ Rs.
- Fc ⁇ Rs For some targets and indications, it may be further beneficial to utilize antibodies that provide differential selectivity for different activating Fc ⁇ Rs; for example, in some cases enhanced binding to Fc ⁇ Rlla and Fc ⁇ Rllla may be desired, but not Fc ⁇ RI, whereas in other cases, enhanced binding only to Fc ⁇ Rlla may be desired.
- Fc ⁇ Rlla Fc ⁇ Rlla
- Fc ⁇ Rllla Fc ⁇ RI
- Fc ⁇ Rlla Fc ⁇ Rlla
- Fc ⁇ Rlla Fc ⁇ Rlla
- complement-mediated effector functions it may be desirable to utilize antibodies that enhance either Fc ⁇ R-mediated or complement-mediated effector functions.
- the target or cancer indications it may be advantageous to reduce or ablate one or more effector functions, for example by knocking out binding to C1q, one or more Fc ⁇ R's, FcRn, or one or more other Fc ligands.
- Fc ligand selectivity or specificity of a given antibody will provide different properties depending on whether it composes an antibody or an antibodies with a coupled fusion or conjugate partner.
- toxin, radionucleotide, or other conjugates may be less toxic to normal cells if the antibody that comprises them has reduced or ablated binding to one or more Fc ligands.
- an antibody that comprises two or more Fc regions with enhanced Fc ⁇ Rllb affinity may co-engage this receptor on the surface of immune cells, thereby inhibiting proliferation of these cells.
- an antibodies may engage its target antigen on one cell type yet engage Fc ⁇ Rs on separate cells from the target antigen, in other cases it may be advantageous to engage Fc ⁇ Rs on the surface of the same cells as the target antigen.
- an antibody targets an antigen on a cell that also expresses one or more Fc ⁇ Rs, it may be beneficial to utilize an antibody that enhances or reduces binding to the Fc ⁇ Rs on the surface of that cell.
- antigen and Fc ⁇ R co-engagement on the same cell may be advantageous when the antibody is being used to modulate the immune system in some way, wherein co- engagement of target antigen and Fc ⁇ R provides some proliferative or anti-proliferative effect.
- antibodies that comprise two or more Fc regions may benefit from antibodies that modulate Fc ⁇ R selectivity or specificity to co-engage Fc ⁇ Rs on the surface of the same cell.
- the Fc ligand specificity of the antibodies of the present invention can be modulated to create different effector function profiles that may be suited for particular antigen epitopes, indications or patient populations.
- Figure 3 describes several embodiments of receptor binding profiles that include improvements to, reductions to or no effect to the binding to various receptors, where such changes may be beneficial in certain contexts.
- the receptor binding profiles in the figure could be varied by degree of increase or decrease to the specified receptors.
- the binding changes specified could be in the context of additional binding changes to other receptors such as C1q or FcRn, for example by combining with ablation of binding to C1q to shut off complement activation, or by combining with enhanced binding to C1q to increase complement activation.
- Other embodiments with other receptor binding profiles are possible, the listed receptor binding profiles are exemplary.
- Antibodies of the present invention may comprise one or more modifications that provide optimized properties that are not specifically related to effector function per se.
- the modifications may be amino acid modifications, or may be modifications that are made enzymatically or chemically. Such modification(s) likely provide some improvement in the antibody, for example an enhancement in its stability, solubility, function, or clinical use.
- variable region of an antibody of the present invention may be affinity matured, that is to say that amino acid modifications have been made in the VH and/or VL domains of the antibody to enhance binding of the antibody to its target antigen.
- Such types of modifications may improve the association and/or the dissociation kinetics for binding to the target antigen.
- Other modifications include those that improve selectivity for target antigen vs. alternative targets. These include modifications that improve selectivity for antigen expressed on target vs. non-target cells.
- Other improvements to the target recognition properties may be provided by additional modifications. Such properties may include, but are not limited to, specific kinetic properties (i.e.
- Antibodies of the invention may comprise one or more modifications that provide reduced or enhanced internalization of an antibody.
- antibodies of the present invention can be utilized or combined with additional modifications in order to reduce the cellular internalization of an antibody that occurs via interaction with one or more Fc ligands. This property might be expected to enhance effector function, and potentially reduce immunogenicity of the antibodies of the invention.
- antibodies of the present antibodies of the present invention can be utilized directly or combined with additional modifications in order to enhance the cellular internalization of an antibody that occurs via interaction with one or more Fc ligands.
- an antibody is used that provides enhanced binding to Fc ⁇ RI, which is expressed on dendritic cells and active early in immune response.
- This strategy could be further enhanced by combination with additional modifications, either within the antibody or in an attached fusion or conjugate partner, that promote recognition and presentation of Fc peptide fragments by MHC molecules.
- idiotype vaccine immunotherapies in which clone-specific antibodies produced by a patient's lymphoma cells are used to vaccinate the patient.
- modifications are made to improve biophysical properties of the antibodies of the present invention, including but not limited to stability, solubility, and oligomeric state.
- Modifications can include, for example, substitutions that provide more favorable intramolecular interactions in the antibody such as to provide greater stability, or substitution of exposed nonpolar amino acids with polar amino acids for higher solubility.
- substitutions that provide more favorable intramolecular interactions in the antibody such as to provide greater stability, or substitution of exposed nonpolar amino acids with polar amino acids for higher solubility.
- a number of optimization goals and methods are described in USSN 10/379,392, incorporated herein it its entirety by reference, that may find use for engineering additional modifications to further optimize the antibodies of the present invention.
- the antibodies of the present invention can also be combined with additional modifications that reduce oligomeric state or size, such that tumor penetration is enhanced, or in vivo clearance rates are increased as desired.
- Other modifications to the antibodies of the present invention include those that enable the specific formation or homodimeric or homomultimeric molecules.
- Such modifications include but are not limited to engineered disulfides, as well as chemical modifications or aggregation methods which may provide a mechanism for generating covalent homodimeric or homomultimers.
- methods of engineering and compositions of such molecules are described in Kan et al., 2001 , J. Immunol., 2001 , 166: 1320-1326; Stevenson et al., 2002, Recent Results Cancer Res. 159: 104-12; US 5,681 ,566; Caron et al., 1992, J. Exp. Med. 176:1191-1195, and Shopes, 1992, J. Immunol. 148(9):2918-22, each incorporated herein it its entirety by reference.
- Additional modifications to the variants of the present invention include those that enable the specific formation or heterodimeric, heteromultimeric, bifunctional, and/or multifunctional molecules.
- modifications include, but are not limited to, one or more amino acid substitutions in the CH3 domain, in which the substitutions reduce homodimer formation and increase heterodimer formation.
- methods of engineering and compositions of such molecules are described in Atwell et al., 1997, J. MoI. Biol. 270(1 ):26-35, and Carter et al., 2001 , J. Immunol. Methods 248:7-15, each incorporated herein it its entirety by reference.
- Additional modifications include modifications in the hinge and CH3 domains, in which the modifications reduce the propensity to form dimers.
- the antibodies of the present invention comprise modifications that remove proteolytic degradation sites. These may include, for example, protease sites that reduce production yields, as well as protease sites that degrade the administered protein in vivo. In one embodiment, additional modifications are made to remove covalent degradation sites such as deamidation (i.e. deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues), oxidation, and proteolytic degradation sites.
- deamidation i.e. deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues
- oxidation oxidation
- Deamidation sites that are particularly useful to remove are those that have enhance propensity for deamidation, including, but not limited to asparaginyl and glutamyl residues followed by glycines (NG and QG motifs, respectively). In such cases, substitution of either residue can significantly reduce the tendency for deamidation. Common oxidation sites include methionine and cysteine residues. Other covalent modifications, that can either be introduced or removed, include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the "-amino groups of lysine, arginine, and histidine side chains (T.E.
- Modifications may include those that improve expression and/or purification yields from hosts or host cells commonly used for production of biologies. These include, but are not limited to various mammalian cell lines (e.g. CHO), yeast cell lines, bacterial cell lines, and plants.
- the antibodies of the present invention may comprise modifications that include the use of unnatural amino acids incorporated using, for example, the technologies developed by Schultz and colleagues, including but not limited to methods described by Cropp & Shultz, 2004, Trends Genet. 20(12):625-30, Anderson et al., 2004, Proc. Natl. Acad. Sci. U.S.A.
- the antibody may be linked to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol.
- PEG polyethylene glycol
- polypropylene glycol polypropylene glycol
- polyoxyalkylenes polypropylene glycol
- Additional amino acid modifications may be made to enable specific or non-specific chemical or posttranslational modification of the antibodies.
- modifications include, but are not limited to PEGylation and glycosylation.
- Specific substitutions that can be utilized to enable PEGylation include, but are not limited to, introduction of novel cysteine residues or unnatural amino acids such that efficient and specific coupling chemistries can be used to attach a PEG or otherwise polymeric moiety.
- Introduction of specific glycosylation sites can be achieved by introducing novel N-X-T/S sequences into the antibodies of the present invention.
- Covalent modifications of antibodies are included within the scope of this invention, and are generally, but not always, done post-translationally.
- the covalent modification of the antibodies of the invention comprises the addition of one or more labels.
- labeling group is any detectable label.
- the labeling group is coupled to the antibody via spacer arms of various lengths to reduce potential steric hindrance.
- Various methods for labeling proteins are known in the art and may be used in performing the present invention.
- labels fall into a variety of classes, depending on the assay in which they are to be detected: a) isotopic labels, which may be radioactive or heavy isotopes; b) magnetic labels (e.g., magnetic particles); c) redox active moieties; d) optical dyes; enzymatic groups (e.g. horseradish peroxidase, ⁇ -galactosidase, luciferase, alkaline phosphatase); e) biotinylated groups; and f) predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags, etc.).
- a secondary reporter e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags, etc.
- the labeling group is coupled to the antibody via spacer arms of various lengths to reduce potential steric hindrance.
- Various methods for labeling proteins are known in the art and may be used in performing the present invention.
- Specific labels include optical dyes, including, but not limited to, chromophores, phosphors and fluorophores, with the latter being specific in many instances.
- Fluorophores can be either "small molecule” fluores, or proteinaceous fluores.
- fluorescent label is meant any molecule that may be detected via its inherent fluorescent properties.
- the antibodies of the invention are antibody "fusion proteins", sometimes referred to herein as "antibody conjugates".
- the fusion partner or conjugate partner can be proteinaceous or non-proteinaceous; the latter generally being generated using functional groups on the antibody and on the conjugate partner.
- Conjugate and fusion partners may be any molecule, including small molecule chemical compounds and polypeptides. For example, a variety of antibody conjugates and methods are described in Trail et al., 1999, Curr. Opin. Immunol. 11 :584-588, incorporated herein it its entirety by reference.
- conjugate partners include but are not limited to cytokines, cytotoxic agents, toxins, radioisotopes, chemotherapeutic agent, anti-angiogenic agents, a tyrosine kinase inhibitors, and other therapeutically active agents.
- conjugate partners may be thought of more as payloads, that is to say that the goal of a conjugate is targeted delivery of the conjugate partner to a targeted cell, for example a cancer cell or immune cell, by the antibody.
- the conjugation of a toxin to an antibody targets the delivery of the toxin to cells expressing the target antigen.
- the concepts and definitions of fusion and conjugate are overlapping.
- an antibody as a fusion or conjugate is not meant to constrain it to any particular embodiment of the present invention. Rather, these terms are used loosely to convey the broad concept that any antibody of the present invention may be linked genetically, chemically, or otherwise, to one or more polypeptides or molecules to provide some desirable property.
- Suitable conjugates include, but are not limited to, labels as described below, drugs and cytotoxic agents including, but not limited to, cytotoxic drugs (e.g., chemotherapeutic agents) or toxins or active fragments of such toxins.
- cytotoxic drugs e.g., chemotherapeutic agents
- Suitable toxins and their corresponding fragments include diptheria A chain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin and the like.
- Cytotoxic agents also include radiochemicals made by conjugating radioisotopes to antibodies, or binding of a radionuclide to a chelating agent that has been covalently attached to the antibody.
- cytokine as used herein is meant a generic term for proteins released by one cell population that act on another cell as intercellular mediators. For example, as described in Penichet et al., 2001 , J. Immunol. Methods 248:91-101 , incorporated herein it its entirety by reference, cytokines may be fused to antibody to provide an array of desirable properties.
- cytokines lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-alpha and -beta; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-beta; platelet-growth factor; transforming growth factors (TGFs) such as TGF-alpha and TGF-beta; insulin
- the antibodies of the present invention are fused, conjugated, or operably linked to a toxin, including but not limited to small molecule toxins and enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof.
- a toxin including but not limited to small molecule toxins and enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof.
- small molecule toxins include but are not limited to calicheamicin, maytansine (US 5,208,020, incorporated herein it its entirety by reference), trichothene, and CC1065.
- the antibody is conjugated to one or more maytansine molecules (e.g. about 1 to about 10 maytansine molecules per antibody molecule).
- Maytansine may, for example, be converted to May-SS-Me which may be reduced to May-SH3 and reacted with modified antibody (Chari et al., 1992, Cancer Research 52: 127-131 , incorporated herein it its entirety by reference) to generate a maytansinoid-antibody conjugate.
- Another conjugate of interest comprises an antibody conjugated to one or more calicheamicin molecules.
- the calicheamicin family of antibiotics are capable of producing double-stranded DNA breaks at sub-picomolar concentrations.
- Structural analogues of calicheamicin that may be used include but are not limited to Y 1 1 , ⁇ 2 1 , ⁇ 3 , N-acetyl-Y ! 1 , PSAG, and ⁇ , (Hinman et al., 1993, Cancer Research 53:3336-3342; Lode et al., 1998, Cancer Research 58:2925-2928) (US 5,714,586; US 5,712,374; US 5,264,586; US 5,773,001 , each incorporated herein it its entirety by reference).
- Dolastatin 10 analogs such as auristatin E (AE) and monomethylauristatin E (MMAE) may find use as conjugates for the antibodies of the present invention (Doronina et al., 2003, Nat Biotechnol 21(7):778-84; Francisco et al., 2003 Blood 102(4): 1458-65, each incorporated herein it its entirety by reference).
- AE auristatin E
- MMAE monomethylauristatin E
- Useful enzymatically active toxins include but are not limited to diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes.
- the present invention further contemplates a conjugate between an antibody of the present invention and a compound with nucleolytic activity, for example a ribonuclease or DNA endonuclease such as a deoxyribonuclease (Dnase).
- a compound with nucleolytic activity for example a ribonuclease or DNA endonuclease such as a deoxyribonuclease (Dnase).
- an antibody of the present invention may be fused, conjugated, or operably linked to a radioisotope to form a radioconjugate.
- a radioactive isotope are available for the production of radioconjugate antibodies. Examples include, but are not limited to, At211 , 1131 , 1125, Y90, Re186, Re188, Sm153, Bi212, P32, and radioactive isotopes of Lu.
- an antibody of the present invention may be conjugated to a "receptor” (such as streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand” (e.g. avidin) which is conjugated to a cytotoxic agent (e.g. a radionucleotide).
- a "ligand” e.g. avidin
- cytotoxic agent e.g. a radionucleotide
- the antibody is conjugated or operably linked to an enzyme in order to employ Antibody Dependent Enzyme Mediated Prodrug Therapy (ADEPT).
- ADPT Antibody Dependent Enzyme Mediated Prodrug Therapy
- ADEPT may be used by conjugating or operably linking the antibody to a prodrug-activating enzyme that converts a prodrug (e.g. a peptidyl chemotherapeutic agent, see PCT WO 81/01145, incorporated herein it its entirety by reference) to an active anti-cancer drug.
- a prodrug e.g. a peptidyl chemotherapeutic agent, see PCT WO 81/01145, incorporated herein it its entirety by reference
- the enzyme component of the immunoconjugate useful for ADEPT includes any enzyme capable of acting on a prodrug in such a way so as to covert it into its more active, cytotoxic form.
- Enzymes that are useful in the method of this invention include but are not limited to alkaline phosphatase useful for converting phosphate-containing prodrugs into free drugs; arylsulfatase useful for converting sulfate-containing prodrugs into free drugs; cytosine deaminase useful for converting non-toxic 5-fluorocytosine into the anti-cancer drug, 5- fluorouracil; proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L), that are useful for converting peptide- containing prodrugs into free drugs; D-alanylcarboxypeptidases, useful for converting prodrugs that contain D-amino acid substituents; carbohydrate-cleaving enzymes such as ⁇ - galactosidase and neuramimidase useful for converting glycosylated prodrugs into free drugs; beta-lactamas
- antibodies with enzymatic activity can be used to convert the prodrugs of the invention into free active drugs (see, for example, Massey, 1987, Nature 328: 457-458, incorporated herein it its entirety by reference).
- Antibody-abzyme conjugates can be prepared for delivery of the abzyme to a tumor cell population.
- additional conjugates are contemplated for the antibodies of the present invention.
- chemotherapeutic agents, anti- angiogenic agents, tyrosine kinase inhibitors, and other therapeutic agents are described below, which may find use as antibody conjugates.
- Fc polypeptides Also contemplated as fusion and conjugate partners are Fc polypeptides.
- an antibody may be a multimeric Fc polypeptide, comprising two or more Fc regions.
- Fc regions may be linked using a chemical engineering approach. For example, Fab's and Fc's may be linked by thioether bonds originating at cysteine residues in the hinges, generating molecules such as FabFc 2 .
- Fc regions may be linked using disulfide engineering and/or chemical cross-linking. In one embodiment, Fc regions may be linked genetically.
- Fc regions in an antibody are linked genetically to generated tandemly linked Fc regions as described in USSN 11/022,289, filed 12/21/2004, entitled "Fc polypeptides with novel Fc ligand binding sites," incorporated herein it its entirety by reference.
- Tandemly linked Fc polypeptides may comprise two or more Fc regions, e.g., one to three, two, etc., Fc regions. It may be advantageous to explore a number of engineering constructs in order to obtain homo- or hetero- tandemly linked antibodies with the most favorable structural and functional properties.
- Tandemly linked antibodies may be homo- tandemly linked antibodies, that is an antibody of one isotype is fused genetically to another antibody of the same isotype.
- antibodies from different isotypes may be tandemly linked, referred to as hetero- tandemly linked antibodies.
- an antibody that binds both Fc ⁇ Rs and Fc ⁇ RI may provide a significant clinical improvement.
- Fc fusion is herein meant to be synonymous with the terms “immunoadhesin”, “Ig fusion”, “Ig chimera”, and “receptor globulin” (sometimes with dashes) as used in the prior art (Chamow et al., 1996, Trends Biotechnol 14:52-60; Ashkenazi et al., 1997, Curr Opin Immunol 9:195-200).
- An Fc fusion is a protein wherein one or more polypeptides is operably linked to Fc.
- An Fc fusion combines the Fc region of an antibody, and thus its favorable effector functions and pharmacokinetics, with the target-binding region of a receptor, ligand, or some other protein or protein domain.
- Protein fusion partners may include, but are not limited to, the variable region of any antibody, the target-binding region of a receptor, an adhesion molecule, a ligand, an enzyme, a cytokine, a chemokine, or some other protein or protein domain. Small molecule fusion partners may include any therapeutic agent that directs the Fc fusion to a therapeutic target.
- Fusion and conjugate partners may be linked to any region of an antibody of the present invention, including at the N- or C- termini, or at some residue in-between the termini.
- a fusion or conjugate partner is linked at the N- or C-terminus of the antibody, e.g., the N-terminus.
- linkers may find use in the present invention to covalently link antibodies to a fusion or conjugate partner.
- linker By “linker”, “linker sequence”, “spacer”, “tethering sequence” or grammatical equivalents thereof, herein is meant a molecule or group of molecules (such as a monomer or polymer) that connects two molecules and often serves to place the two molecules in a desirable configuration.
- Linkers are known in the art; for example, homo-or hetero-bifunctional linkers as are well known (see, 1994 Pierce Chemical Company catalog, technical section on cross-linkers, pages 155-200, incorporated herein it its entirety by reference). A number of strategies may be used to covalently link molecules together.
- the linker is a peptide bond, generated by recombinant techniques or peptide synthesis.
- the linker may contain amino acid residues that provide flexibility.
- the linker peptide may predominantly include the following amino acid residues: GIy, Ser, Ala, or Thr.
- the linker peptide should have a length that is adequate to link two molecules in such a way that they assume the correct conformation relative to one another so that they retain the desired activity. Suitable lengths for this purpose include at least one and not more than 50 amino acid residues.
- the linker is from about 1 to 30 amino acids in length, with linkers of 1 to 20 amino acids in length being desirable.
- Useful linkers include glycine-serine polymers (including, for example, (GS)n, (GSGGS)n (GGGGS)n and (GGGS)n, where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers, as will be appreciated by those in the art.
- nonproteinaceous polymers including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol, may find use as linkers, that is may find use to link the antibodies of the present invention to a fusion or conjugate partner, or to link the antibodies of the present invention to a conjugate.
- PEG polyethylene glycol
- polypropylene glycol polypropylene glycol
- polyoxyalkylenes polyoxyalkylenes
- copolymers of polyethylene glycol and polypropylene glycol may find use as linkers, that is may find use to link the antibodies of the present invention to a fusion or conjugate partner, or to link the antibodies of the present invention to a conjugate.
- the present invention provides methods for producing and experimentally testing antibodies.
- the described methods are not meant to constrain the present invention to any particular application or theory of operation. Rather, the provided methods are meant to illustrate generally that one or more antibodies may be produced and experimentally tested to obtain variant antibodies.
- nucleic acids are created that encode the antibodies, and that may then be cloned into host cells, expressed and assayed, if desired.
- nucleic acids may be made that encode each protein sequence.
- These practices are carried out using well-known procedures. For example, a variety of methods that may find use in the present invention are described in Molecular Cloning - A Laboratory Manual, 3 rd Ed. (Maniatis, Cold Spring Harbor Laboratory Press, New York, 2001 ), and Current Protocols in Molecular Biology (John Wiley & Sons), each incorporated herein it its entirety by reference.
- the generation of exact sequences for a library comprising a large number of sequences is potentially expensive and time consuming.
- library herein is meant a set of variants in any form, including but not limited to a list of nucleic acid or amino acid sequences, a list of nucleic acid or amino acid substitutions at variable positions, a physical library comprising nucleic acids that encode the library sequences, or a physical library comprising the variant proteins, either in purified or unpurified form. Accordingly, there are a variety of techniques that may be used to efficiently generate libraries of the present invention.
- Such methods that may find use in the present invention are described or referenced in US 6,403,312; USSN 09/782,004; USSN 09/927,790; USSN 10/218,102; PCT WO 01/40091 ; and PCT WO 02/25588, each incorporated herein it its entirety by reference.
- Such methods include but are not limited to gene assembly methods, PCR-based method and methods which use variations of PCR, ligase chain reaction-based methods, pooled oligo methods such as those used in synthetic shuffling, error-prone amplification methods and methods which use oligos with random mutations, classical site-directed mutagenesis methods, cassette mutagenesis, and other amplification and gene synthesis methods.
- the antibodies of the present invention may be produced by culturing a host cell transformed with nucleic acid, e.g., an expression vector, containing nucleic acid encoding the antibodies, under the appropriate conditions to induce or cause expression of the protein.
- nucleic acid e.g., an expression vector
- the conditions appropriate for expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation.
- a wide variety of appropriate host cells may be used, including but not limited to mammalian cells, bacteria, insect cells, and yeast.
- a variety of cell lines that may find use in the present invention are described in the ATCC® cell line catalog, available from the American Type Culture Collection.
- the antibodies are expressed in mammalian expression systems, including systems in which the expression constructs are introduced into the mammalian cells using virus such as retrovirus or adenovirus.
- virus such as retrovirus or adenovirus.
- Any mammalian cells may be used, e.g., human, mouse, rat, hamster, primate cells, etc.. Suitable cells also include known research cells, including but not limited to Jurkat T cells, NIH3T3, CHO, BHK, COS, HEK293, PER C.6, HeLa, Sp2/0, NSO cells and variants thereof.
- library proteins are expressed in bacterial cells.
- Bacterial expression systems are well known in the art, and include Escherichia coli (E.
- antibodies are produced in insect cells (e.g. Sf21/Sf9, Trichoplusia ni Bti-Tn5b1-4) or yeast cells (e.g. S. cerevisiae, Pichia, etc).
- insect cells e.g. Sf21/Sf9, Trichoplusia ni Bti-Tn5b1-4
- yeast cells e.g. S. cerevisiae, Pichia, etc.
- antibodies are expressed in vitro using cell free translation systems. In vitro translation systems derived from both prokaryotic (e.g. E. coli) and eukaryotic (e.g. wheat germ, rabbit reticulocytes) cells are available and may be chosen based on the expression levels and functional properties of the protein of interest.
- in vitro translation is required for some display technologies, for example ribosome display.
- the antibodies may be produced by chemical synthesis methods.
- transgenic expression systems both animal (e.g. cow, sheep or goat milk, embryonated hen's eggs, whole insect larvae, etc.) and plant (e.g. corn, tobacco, duckweed, etc.)
- the nucleic acids that encode the antibodies of the present invention may be incorporated into an expression vector in order to express the protein.
- a variety of expression vectors may be utilized for protein expression.
- Expression vectors may comprise self-replicating extra-chromosomal vectors or vectors which integrate into a host genome. Expression vectors are constructed to be compatible with the host cell type.
- expression vectors which find use in the present invention include but are not limited to those which enable protein expression in mammalian cells, bacteria, insect cells, yeast, and in in vitro systems.
- a variety of expression vectors are available, commercially or otherwise, that may find use in the present invention for expressing antibodies.
- Expression vectors typically comprise a protein operably linked with control or regulatory sequences, selectable markers, any fusion partners, and/or additional elements.
- operably linked herein is meant that the nucleic acid is placed into a functional relationship with another nucleic acid sequence.
- these expression vectors include transcriptional and translational regulatory nucleic acid operably linked to the nucleic acid encoding the antibody, and are typically appropriate to the host cell used to express the protein.
- the transcriptional and translational regulatory sequences may include promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences.
- expression vectors typically contain a selection gene or marker to allow the selection of transformed host cells containing the expression vector. Selection genes are well known in the art and will vary with the host cell used.
- Antibodies may be operably linked to a fusion partner to enable targeting of the expressed protein, purification, screening, display, and the like. Fusion partners may be linked to the antibody sequence via a linker sequence.
- the linker sequence will generally comprise a small number of amino acids, typically less than ten, although longer linkers may also be used. Typically, linker sequences are selected to be flexible and resistant to degradation. As will be appreciated by those skilled in the art, any of a wide variety of sequences may be used as linkers.
- a common linker sequence comprises the amino acid sequence GGGGS.
- a fusion partner may be a targeting or signal sequence that directs antibody and any associated fusion partners to a desired cellular location or to the extracellular media. As is known in the art, certain signaling sequences may target a protein to be either secreted into the growth media, or into the periplasmic space, located between the inner and outer membrane of the cell.
- a fusion partner may also be a sequence that encodes a peptide or protein that enables purification and/or screening.
- Such fusion partners include but are not limited to polyhistidine tags (His-tags) (for example H 6 and H 10 or other tags for use with Immobilized Metal Affinity Chromatography (IMAC) systems (e.g.
- tags which are targeted by antibodies (for example c-myc tags, flag-tags, and the like).
- tags may be useful for purification, for screening, or both.
- an antibody may be purified using a His-tag by immobilizing it to a Ni +2 affinity column, and then after purification the same His-tag may be used to immobilize the antibody to a Ni +2 coated plate to perform an ELISA or other binding assay (as described below).
- a fusion partner may enable the use of a selection method to screen antibodies (see below).
- Fusion partners that enable a variety of selection methods are well-known in the art, and all of these find use in the present invention. For example, by fusing the members of an antibody library to the gene III protein, phage display can be employed (Kay et al., Phage display of peptides and proteins: a laboratory manual, Academic Press, San Diego, CA, 1996; Lowman et al., 1991 , Biochemistry 30:10832-10838; Smith, 1985, Science 228:1315-1317, incorporated herein it its entirety by reference). Fusion partners may enable antibodies to be labeled.
- a fusion partner may bind to a specific sequence on the expression vector, enabling the fusion partner and associated antibody to be linked covalently or noncovalently with the nucleic acid that encodes them.
- the methods of introducing exogenous nucleic acid into host cells are well known in the art, and will vary with the host cell used. Techniques include but are not limited to dextran-mediated transfection, calcium phosphate precipitation, calcium chloride treatment, polybrene mediated transfection, protoplast fusion, electroporation, viral or phage infection, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.
- transfection may be either transient or stable.
- antibodies are purified or isolated after expression. Proteins may be isolated or purified in a variety of ways known to those skilled in the art. Standard purification methods include chromatographic techniques, including ion exchange, hydrophobic interaction, affinity, sizing or gel filtration, and reversed-phase, carried out at atmospheric pressure or at high pressure using systems such as FPLC and HPLC. Purification methods also include electrophoretic, immunological, precipitation, dialysis, and chromatofocusing techniques. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful.
- the bacterial proteins A and G bind to the Fc region.
- the bacterial protein L binds to the Fab region of some antibodies, as of course does the antibody's target antigen. Purification can often be enabled by a particular fusion partner. For example, antibodies may be purified using glutathione resin if a GST fusion is employed, Ni +2 affinity chromatography if a His-tag is employed, or immobilized anti-flag antibody if a flag-tag is used. For general guidance in suitable purification techniques, see, e.g.
- Protein Purification Principles and Practice, 3 rd Ed., Scopes, Springer- Verlag, NY, 1994, incorporated herein it its entirety by reference.
- the degree of purification necessary will vary depending on the screen or use of the antibodies. In some instances no purification is necessary.
- screening may take place directly from the media. As is well known in the art, some methods of selection do not involve purification of proteins. Thus, for example, if a library of antibodies is made into a phage display library, protein purification may not be performed.
- Antibodies may be screened using a variety of methods, including but not limited to those that use in vitro assays, in vivo and cell-based assays, and selection technologies. Automation and high-throughput screening technologies may be utilized in the screening procedures. Screening may employ the use of a fusion partner or label. The use of fusion partners has been discussed above.
- label herein is meant that the antibodies of the invention have one or more elements, isotopes, or chemical compounds attached to enable the detection in a screen.
- labels fall into three classes: a) immune labels, which may be an epitope incorporated as a fusion partner that is recognized by an antibody, b) isotopic labels, which may be radioactive or heavy isotopes, and c) small molecule labels, which may include fluorescent and colorimetric dyes, or molecules such as biotin that enable other labeling methods. Labels may be incorporated into the compound at any position and may be incorporated in vitro or in vivo during protein expression. [203] In one embodiment, the functional and/or biophysical properties of antibodies are screened in an in vitro assay. In vitro assays may allow a broad dynamic range for screening properties of interest.
- the screen is a qualitative or quantitative binding assay for binding of antibodies to a protein or nonprotein molecule that is known or thought to bind the antibody.
- the screen is a binding assay for measuring binding to the target antigen.
- the screen is an assay for binding of antibodies to an Fc ligand, including but not limited to the family of Fc ⁇ Rs, the neonatal receptor FcRn, the complement protein C1q, and the bacterial proteins A and G.
- the Fc ligands may be from any organism, e.g., humans, mice, rats, rabbits, monkeys, etc.. Binding assays can be carried out using a variety of methods known in the art, including but not limited to FRET (Fluorescence Resonance Energy Transfer) and BRET (Bioluminescence Resonance Energy Transfer) -based assays, AlphaScreenTM (Amplified Luminescent Proximity Homogeneous Assay), Scintillation Proximity Assay, ELISA (Enzyme-Linked Immunosorbent Assay), SPR (Surface Plasmon Resonance, also known as BiacoreTM), isothermal titration calorimetry, differential scanning calorimetry, gel electrophoresis, and chromatography including gel filtration. These and other methods may take advantage of some fusion partner or label of the antibody. Assays may employ a variety of detection methods including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels
- the biophysical properties of antibodies may be screened using a variety of methods known in the art.
- Protein stability may be determined by measuring the thermodynamic equilibrium between folded and unfolded states.
- antibodies of the present invention may be unfolded using chemical denaturant, heat, or pH, and this transition may be monitored using methods including but not limited to circular dichroism spectroscopy, fluorescence spectroscopy, absorbance spectroscopy, NMR spectroscopy, calorimetry, and proteolysis.
- the kinetic parameters of the folding and unfolding transitions may also be monitored using these and other techniques.
- the solubility and overall structural integrity of an antibody may be quantitatively or qualitatively determined using a wide range of methods that are known in the art.
- Methods which may find use in the present invention for characterizing the biophysical properties of antibodies include gel electrophoresis, isoelectric focusing, capillary electrophoresis, chromatography such as size exclusion chromatography, ion-exchange chromatography, and reversed-phase high performance liquid chromatography, peptide mapping, oligosaccharide mapping, mass spectrometry, ultraviolet absorbance spectroscopy, fluorescence spectroscopy, circular dichroism spectroscopy, isothermal titration calorimetry, differential scanning calorimetry, analytical ultra-centrifugation, dynamic light scattering, proteolysis, and cross-linking, turbidity measurement, filter retardation assays, immunological assays, fluorescent dye binding assays, protein-staining assays, microscopy, and detection of aggregates via ELISA or other binding assay.
- Structural analysis employing X-ray crystallographic techniques and NMR spectroscopy may also find use.
- stability and/or solubility may be measured by determining the amount of protein solution after some defined period of time.
- the protein may or may not be exposed to some extreme condition, for example elevated temperature, low pH, or the presence of denaturant.
- the aforementioned functional and binding assays also provide ways to perform such a measurement. For example, a solution comprising an antibody could be assayed for its ability to bind target antigen, then exposed to elevated temperature for one or more defined periods of time, then assayed for antigen binding again.
- the library is screened using one or more cell-based or in vitro assays.
- antibodies, purified or unpurified are typically added exogenously such that cells are exposed to individual variants or groups of variants belonging to a library.
- These assays are typically, but not always, based on the biology of the ability of the antibody to bind to antigen and mediate some biochemical event, for example effector functions like cellular lysis, phagocytosis, ligand/receptor binding inhibition, inhibition of growth and/or proliferation, apoptosis, etc.
- Such assays often involve monitoring the response of cells to antibody, for example cell survival, cell death, cellular phagocytosis, cell lysis, change in cellular morphology, or transcriptional activation such as cellular expression of a natural gene or reporter gene.
- such assays may measure the ability of antibodies to elicit ADCC, ADCP, or CDC.
- additional cells or components that is in addition to the target cells, may need to be added, for example serum complement, or effector cells such as peripheral blood monocytes (PBMCs), NK cells, macrophages, and the like.
- PBMCs peripheral blood monocytes
- NK cells macrophages, and the like.
- additional cells may be from any organism, e.g., humans, mice, rats, rabbits, monkeys, etc.
- Crosslinked or monomeric antibodies may cause apoptosis of certain cell lines expressing the antibody's target antigen, or they may mediate attack on target cells by immune cells which have been added to the assay.
- Methods for monitoring cell death or viability include the use of dyes, fluorophores, immunochemical, cytochemical, and radioactive reagents.
- caspase assays or annexin- flourconjugates may enable apoptosis to be measured, and uptake or release of radioactive substrates (e.g. Chromium-51 release assays) or the metabolic reduction of fluorescent dyes such as alamar blue may enable cell growth, proliferation, or activation to be monitored.
- the DELFIA® EuTDA-based cytotoxicity assay (Perkin Elmer, MA) is used.
- dead or damaged target cells may be monitored by measuring the release of one or more natural intracellular proteins, for example lactate dehydrogenase.
- Transcriptional activation may also serve as a method for assaying function in cell-based assays.
- response may be monitored by assaying for natural genes or proteins which may be upregulated or down-regulated, for example the release of certain interleukins may be measured, or alternatively readout may be via a luciferase or GFP-reporter construct.
- Cell-based assays may also involve the measure of morphological changes of cells as a response to the presence of an antibody.
- Cell types for such assays may be prokaryotic or eukaryotic, and a variety of cell lines that are known in the art may be employed. Alternatively, cell-based screens are performed using cells that have been transformed or transfected with nucleic acids encoding the antibodies.
- In vitro assays include but are not limited to binding assays, ADCC, CDC, phagocytosis, cytotoxicity, proliferation, apoptosis, necrosis, cell cycle arrest, peroxide/ozone release, chemotaxis of effector cells, inhibition of such assays by reduced effector function antibodies; ranges of activities such as >100x improvement or >100x reduction, blends of receptor activation and the assay outcomes that are expected from such receptor profiles.
- the biological properties of the antibodies of the present invention may be characterized in cell, tissue, and whole organism experiments.
- drugs are often tested in animals, including but not limited to mice, rats, rabbits, dogs, cats, pigs, and monkeys, in order to measure a drug's efficacy for treatment against a disease or disease model, or to measure a drug's pharmacokinetics, toxicity, and other properties.
- the animals may be referred to as disease models.
- Therapeutics are often tested in mice, including but not limited to nude mice, SCID mice, xenograft mice, and transgenic mice (including knockins and knockouts).
- an antibody of the present invention that is intended as an anti-cancer therapeutic may be tested in a mouse cancer model, for example a xenograft mouse.
- a tumor or tumor cell line is grafted onto or injected into a mouse, and subsequently the mouse is treated with the therapeutic to determine the ability of the antibody to reduce or inhibit cancer growth and metastasis.
- An alternative approach is the use of a SCID murine model in which immune-deficient mice are injected with human Periferal Blood Lymphocytes (PBLs), conferring a semi-functional and human immune system - with an appropriate array of human FcRs - to the mice that have subsequently been injected with antibodies or Fc-polypeptides that target injected human tumor cells.
- PBLs Periferal Blood Lymphocytes
- the Fc-polypeptides that target the desired antigen such as her2/neu on SkOV3 ovarian cancer cells
- Such experimentation may provide meaningful data for determination of the potential of the antibody to be used as a therapeutic. Any organism, e.g., mammals, may be used for testing.
- monkeys can be suitable therapeutic models, and thus may be used to test the efficacy, toxicity, pharmacokinetics, or other property of the antibodies of the present invention.
- Tests of the antibodies of the present invention in humans are ultimately required for approval as drugs, and thus of course these experiments are contemplated.
- the antibodies of the present invention may be tested in humans to determine their therapeutic efficacy, toxicity, pharmacokinetics, and/or other clinical properties.
- the antibodies of the present invention may confer superior performance on Fc- containing therapeutics in animal models or in humans.
- the receptor binding profiles of such antibodies may, for example, be selected to increase the potency of cytotoxic drugs or to target specific effector functions or effector cells to improve the selectivity of the drug's action. Further, receptor binding profiles can be selected that may reduce some or all effector functions thereby reducing the side-effects or toxicity of such Fc-containing drug. For example, an antibody with reduced binding to Fc ⁇ Rllla, Fc ⁇ RI and Fc ⁇ Rlla can be selected to eliminate most cell-mediated effector function, or an antibody with reduced binding to C1q may be selected to limit complement-mediated effector functions.
- effector functions are known to have potential toxic effects, therefore eliminating them may increase the safety of the Fc-bearing drug and such improved safety may be characterized in animal models.
- effector functions are known to mediate the desirable therapeutic activity, therefore enhancing them may increase the activity or potency of the Fc-bearing drug and such improved activity or potency may be characterized in animal models.
- Optimized antibodies can be tested in a variety of orthotopic tumor models. These clinically relevant animal models are important in the study of pathophysiology and therapy of aggressive cancers like pancreatic, prostate and breast cancer. Immune deprived mice including, but not limited to athymic nude or SCID mice are frequently used in scoring of local and systemic tumor spread from the site of intraorgan (e.g. pancreas, prostate or mammary gland) injection of human tumor cells or fragments of donor patients. [211] In some embodiments, antibodies of the present invention may be assessed for efficacy in clinically relevant animal models of various human diseases. In many cases, relevant models include various transgenic animals for specific tumor antigens.
- Relevant transgenic models such as those that express human Fc receptors (e.g., CD16 including the gamma chain, Fc ⁇ RI , Rlla/b, and others) could be used to evaluate and test antibodies and Fc-fusions in their efficacy.
- the evaluation of antibodies by the introduction of human genes that directly or indirectly mediate effector function in mice or other rodents may enable physiological studies of efficacy in tumor toxicity or other diseases such as autoimmune disorders and RA.
- Human Fc receptors such as Fc ⁇ Rllla may possess polymorphisms such as that in position 158 V or F which would further enable the introduction of specific and combinations of human polymorphisms into rodents.
- proxy molecules may mimic - in the animal system - the FcR and/or complement biology of a corresponding candidate human antibody. This mimicry is most likely to be manifested by relative association affinities between specific antibodies and animal vs. human receptors. For example, if one were using a mouse model to assess the potential in-human efficacy of an antibody that has enhanced affinity for human Fc ⁇ Rllla, an appropriate proxy variant would have enhanced affinity for mouse Fc ⁇ RIII-2 (mouse CD16-2).
- proxy antibodies could be created in the context of a human antibody, an animal antibody, or both.
- the testing of antibodies may include study of efficacy in primates (e.g. cynomolgus monkey model) to facilitate the evaluation of depletion of specific target cells harboring target antigen.
- primates e.g. cynomolgus monkey model
- Additional primate models include but are not limited to that of the rhesus monkey and Fc polypeptides in therapeutic studies of autoimmune, transplantation, and cancer.
- Toxicity studies are performed to determine the antibody or Fc-fusion related-effects that cannot be evaluated in standard pharmacology profile or occur only after repeated administration of the agent. Most toxicity tests are performed in two species - a rodent and a non-rodent - to ensure that any unexpected adverse effects are not overlooked before new therapeutic entities are introduced into man. In general, these models may measure a variety of toxicities including genotoxicity, chronic toxicity, immunogenicity, reproductive/developmental toxicity, and carcinogenicity. Included within the aforementioned parameters are standard measurement of food consumption, bodyweight, antibody formation, clinical chemistry, and macro- and microscopic examination of standard organs/tissues (e.g. cardiotoxicity).
- PK pharmacokinetics
- the pharmacokinetics (PK) of the antibodies of the invention can be studied in a variety of animal systems, with the most relevant being non-human primates such as the cynomolgus, rhesus monkeys.
- Single or repeated i.v./s.c. administrations over a dose range of 6000-fold (0.05-300 mg/kg) can be evaluated for the half-life (days to weeks) using plasma concentration and clearance as well as volume of distribution at a steady state and level of systemic absorbance can be measured.
- Examples of such parameters of measurement generally include maximum observed plasma concentration (Cmax), the time to reach Cmax (Tmax), the area under the plasma concentration-time curve from time 0 to infinity [AUC(0-inf] and apparent elimination half-life (T 1/2 ). Additional measured parameters could include compartmental analysis of concentration-time data obtained following i.v. administration and bioavailability. Examples of pharmacological/toxicological studies using cynomolgus have been established for Rituxan ® and Zevalin ® in which monoclonal antibodies to CD20 are cross-reactive. Biodistribution, dosimetry (for radiolabled antibodies), and PK studies can also be done in rodent models.
- the antibodies of the present invention may confer superior pharmacokinetics on Fc- containing therapeutics in animal systems or in humans. For example, increased binding to FcRn may increase the half-life and exposure of the Fc-containing drug. Alternatively, decreased binding to FcRn may decrease the half-life and exposure of the Fc-containing drug in cases where reduced exposure is favorable such as when such drug has side- effects.
- Pharmacodynamic studies may include, but are not limited to, targeting specific tumor cells or blocking signaling mechanisms, measuring depletion of target antigen expressing cells or signals, etc.
- the antibodies of the present invention may target particular effector cell populations and thereby direct Fc-containing drugs to recruit certain activities to improve potency or to increase penetration into a particularly favorable physiological compartment.
- neutrophil activity and localization can be targeted, e.g., by an antibody that targets Fc ⁇ Rlllb.
- Such pharmacodynamic effects may be demonstrated in animal models or in humans.
- the antibodies of the present invention may be used for various therapeutic purposes. As will be appreciated by those in the art, the antibodies of the present invention may be used for any therapeutic purpose that uses antibodies and the like. In one embodiment, the antibodies are administered to a patient to treat disorders including but not limited to cancer, autoimmune and inflammatory diseases, and infectious diseases.
- a "patient” for the purposes of the present invention includes both humans and other animals, e.g., mammals, e.g., humans.
- the antibodies of the present invention have both human therapy and veterinary applications.
- treatment or “treating” in the present invention is meant to include therapeutic treatment, as well as prophylactic, or suppressive measures for a disease or disorder.
- successful administration of an antibody prior to onset of the disease results in treatment of the disease.
- successful administration of an optimized antibody after clinical manifestation of the disease to combat the symptoms of the disease comprises treatment of the disease.
- Treatment and “treating” also encompasses administration of an optimized antibody after the appearance of the disease in order to eradicate the disease.
- Successful administration of an agent after onset and after clinical symptoms have developed, with possible abatement of clinical symptoms and perhaps amelioration of the disease, comprises treatment of the disease.
- Those "in need of treatment” include mammals already having the disease or disorder, as well as those prone to having the disease or disorder, including those in which the disease or disorder is to be prevented.
- an antibody of the present invention is administered to a patient having a disease involving inappropriate expression of a protein or other molecule.
- this is meant to include diseases and disorders characterized by aberrant proteins, due for example to alterations in the amount of a protein present, protein localization, posttranslational modification, conformational state, the presence of a mutant or pathogen protein, etc.
- the disease or disorder may be characterized by alterations molecules including but not limited to polysaccharides and gangliosides.
- An overabundance may be due to any cause, including but not limited to overexpression at the molecular level, prolonged or accumulated appearance at the site of action, or increased activity of a protein relative to normal.
- diseases and disorders characterized by a reduction of a protein.
- This reduction may be due to any cause, including but not limited to reduced expression at the molecular level, shortened or reduced appearance at the site of action, mutant forms of a protein, or decreased activity of a protein relative to normal.
- Such an overabundance or reduction of a protein can be measured relative to normal expression, appearance, or activity of a protein, and the measurement may play an important role in the development and/or clinical testing of the antibodies of the present invention.
- cancer and “cancerous” herein refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
- examples of cancer include but are not limited to carcinoma, lymphoma, blastoma, sarcoma (including liposarcoma), neuroendocrine tumors, mesothelioma, schwanoma, meningioma, adenocarcinoma, melanoma, and leukemia or lymphoid malignancies.
- NHL cancers include but are not limited to Burkitt's lymphoma (BL), small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL), mantle cell lymphoma (MCL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLCL), marginal zone lymphoma (MZL), hairy cell leukemia (HCL) and lymphoplasmacytic leukemia (LPL), extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT), nodal marginal zone B cell lymphoma, mediastinal large cell lymphoma, intravascular large cell lymphoma, primary effusion lymphoma, precursor B-lymphoblastic leukemia/lymphoma, precursor T- and NK-cells lymphoma
- BL Burkitt's lymphoma
- SLL/CLL small lymphocytic lymphoma/chronic lympho
- IA are not limited to Hodgkin's lymphoma, tumors of lymphocyte precursor cells, including B- cell acute lymphoblastic leukemia/lymphoma (B-ALL), and T-cell acute lymphoblastic leukemia/lymphoma (T-ALL), thymoma, Langerhans cell histocytosis, multiple myeloma (MM), myeloid neoplasias such as acute myelogenous leukemias (AML), including AML with maturation, AML without differentiation, acute promyelocytic leukemia, acute myelomonocytic leukemia, and acute monocytic leukemias, myelodysplastic syndromes, and chronic myeloproliferative disorders (MDS), including chronic myelogenous leukemia (CML).
- B-ALL B- cell acute lymphoblastic leukemia/lymphoma
- T-ALL T-cell acute lymphoblastic leukemia/lymphoma
- cancers that may be treatable by the antibodies of the invention include but are not limited to tumors of the central nervous system such as glioma, glioblastoma, neuroblastoma, astrocytoma, medulloblastoma, ependymoma, and retinoblastoma; solid tumors of the head and neck (e.g. nasopharyngeal cancer, salivary gland carcinoma, and esophageal cancer), lung (eg. small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung), digestive system (eg.
- gastric or stomach cancer including gastrointestinal cancer, cancer of the bile duct or biliary tract, colon cancer, rectal cancer, colorectal cancer, and anal carcinoma
- reproductive system eg. testicular, penile, or prostate cancer, uterine, vaginal, vulval, cervical, ovarian, and endometrial cancer
- skin eg. melanoma, basal cell carcinoma, squamous cell cancer, actinic keratosis
- liver eg. liver cancer, hepatic carcinoma, hepatocellular cancer, and hepatoma
- bone eg. osteoclastoma, and osteolytic bone cancers
- additional tissues and organs eg.
- pancreatic cancer bladder cancer, kidney or renal cancer, thyroid cancer, breast cancer, cancer of the peritoneum, and Kaposi's sarcoma), and tumors of the vascular system (eg. angiosarcoma and hemagiopericytoma).
- tumors of the vascular system eg. angiosarcoma and hemagiopericytoma.
- Indications that may be treated by anti-CD40 antibodies of the invention include but are not limited to all non-Hodgkin's lymphomas (NHL), especially refractory/resistant NHL, chronic lymphocytic leukemia (CLL), B-cell acute lymphoblastic leukemia/lymphoma (B- ALL), mantle cell lymphoma (MCL), and multiple myeloma (MM).
- NHL non-Hodgkin's lymphomas
- NHL non-Hodgkin's lymphomas
- NHL chronic lymphocytic leukemia
- B- ALL B-cell acute lymphoblastic leukemia/lymphoma
- MCL mantle cell lymphoma
- MM multiple myeloma
- autoimmune diseases include allogenic islet graft rejection, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, antineutrophil cytoplasmic autoantibodies (ANCA), autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune myocarditis, autoimmune neutropenia, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, autoimmune urticaria, Behcet's disease, bullous pemphigoid, cardiomyopathy, Castleman's syndrome, celiac spruce-dermatitis, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, dermatomyositis, discoid lupus, essential
- inflammatory disorders include acute respiratory distress syndrome (ARDS), acute septic arthritis, adjuvant arthritis, juvenile idiopathic arthritis, allergic encephalomyelitis, allergic rhinitis, allergic vasculitis, allergy, asthma, atherosclerosis, chronic inflammation due to chronic bacterial or viral infections, chronic obstructive pulmonary disease (COPD), coronary artery disease, encephalitis, inflammatory bowel disease, inflammatory osteolysis, inflammation associated with acute and delayed hypersensitivity reactions, inflammation associated with tumors, peripheral nerve injury or demyelinating diseases, inflammation associated with tissue trauma such as burns and ischemia, inflammation due to meningitis, multiple organ injury syndrome, pulmonary fibrosis, sepsis and septic shock, Stevens-Johnson syndrome, undifferentiated arthropy, and undifferentiated spondyloarthropathy.
- ARDS acute respiratory distress syndrome
- acute septic arthritis adjuvant arthritis
- juvenile idiopathic arthritis juvenile idiopathic arthritis
- infectious diseases include diseases caused by pathogens such as viruses, bacteria, fungi, protozoa, and parasites. Infectious diseases may be caused by viruses including adenovirus, cytomegalovirus, dengue, Epstein-Barr, hanta, hepatitis A, hepatitis B, hepatitis C, herpes simplex type I, herpes simplex type II, human immunodeficiency virus (HIV), human papilloma virus (HPV), influenza, measles, mumps, papova virus, polio, respiratory syncytial virus (RSV), rinderpest, rhinovirus, rotavirus, rubella, SARS virus, smallpox, viral meningitis, and the like.
- viruses including adenovirus, cytomegalovirus, dengue, Epstein-Barr, hanta, hepatitis A, hepatitis B, hepatitis C, herpes simplex type I, her
- Infections diseases may also be caused by bacteria including Bacillus anthracis, Borrelia burgdorferi, Campylobacter jejuni, Chlamydia trachomatis, Clostridium botulinum, Clostridium tetani, Diptheria, E. coli, Legionella, Helicobacter pylori, Mycobacterium rickettsia, Mycoplasma nesisseria, Pertussis, Pseudomonas aeruginosa, S. pneumonia, Streptococcus, Staphylococcus, Vibria cholerae, Yersinia pestis, and the like.
- bacteria including Bacillus anthracis, Borrelia burgdorferi, Campylobacter jejuni, Chlamydia trachomatis, Clostridium botulinum, Clostridium tetani, Diptheria, E. coli, Legionella, Helicobacter pylori, Mycobacter
- Infectious diseases may also be caused by fungi such as Aspergillus fumigatus, Blastomyces dermatitidis, Candida albicans, Coccidioides immitis, Cryptococcus neoformans, Histoplasma capsulatum, Penicillium mameffei, and the like. Infectious diseases may also be caused by protozoa and parasites such as chlamydia, kokzidioa, leishmania, malaria, rickettsia, trypanosoma, and the like.
- antibodies of the present invention may be used to prevent or treat additional conditions including but not limited to heart conditions such as congestive heart failure (CHF), myocarditis and other conditions of the myocardium; skin conditions such as rosecea, acne, and eczema; bone and tooth conditions such as bone loss, osteoporosis, Paget's disease, Langerhans' cell histiocytosis, periodontal disease, disuse osteopenia, osteomalacia, monostotic fibrous dysplasia, polyostotic fibrous dysplasia, bone metastasis, bone pain management, humoral malignant hypercalcemia, periodontal reconstruction, spinal cord injury, and bone fractures; metabolic conditions such as Gaucher's disease; endocrine conditions such as Cushing's syndrome; and neurological conditions.
- CHF congestive heart failure
- myocarditis and other conditions of the myocardium skin conditions such as rosecea, acne, and eczema
- bone and tooth conditions such as bone loss, osteoporosis, Page
- a number of the receptors that may interact with the antibodies of the present invention are polymorphic in the human population.
- the efficacy of the antibodies of the present invention may be affected by the presence or absence of specific polymorphisms in proteins.
- Fc ⁇ RIIIA is polymorphic at position 158, which is commonly either V (high affinity) or F (low affinity).
- Additional polymorphisms include but are not limited to Fc ⁇ RIIA R131 or H131 , and such polymorphisms are known to either increase or decrease Fc binding and subsequent biological activity, depending on the polymorphism, antibodies of the present invention may bind to a particular polymorphic form of a receptor, for example Fc ⁇ RIIIA 158 V, or to bind with equivalent affinity to all of the polymorphisms at a particular position in the receptor, for example both the 158V and 158F polymorphisms of Fc ⁇ RIIIA. In one embodiment, antibodies of the present invention may have equivalent binding to polymorphisms that may be used in an antibody to eliminate the differential efficacy seen in patients with different polymorphisms.
- Such a property may give greater consistency in therapeutic response and reduce non-responding patient populations.
- Such variant Fc with identical binding to receptor polymorphisms may have increased biological activity, such as ADCC, CDC or circulating half-life, or alternatively decreased activity, via modulation of the binding to the relevant Fc receptors.
- antibodies of the present invention may bind with higher or lower affinity to one of the polymorphisms of a receptor, either accentuating the existing difference in binding or reversing the difference.
- Such a property may allow creation of therapeutics particularly tailored for efficacy with a patient population possessing such polymorphism.
- a patient population possessing a polymorphism with a higher affinity for an inhibitory receptor such as Fc ⁇ RIIB could receive a drug containing an antibody with reduced binding to such polymorphic form of the receptor, creating a more efficacious drug.
- patients are screened for one or more polymorphisms in order to predict the efficacy of the antibodies of the present invention.
- This information may be used, for example, to select patients to include or exclude from clinical trials or, post-approval, to provide guidance to physicians and patients regarding appropriate dosages and treatment options.
- Fc ⁇ RIIIA 158F antibody drugs such as the anti-CD20 mAb
- Rituxan ® are minimally effective (Carton 2002 Blood 99: 754-758; Weng 2003 J. Clin. Oncol. 21 :3940-3947, each incorporated herein it its entirety by reference); such patients may show a much better clinical response to the antibodies of the present invention.
- patients are selected for inclusion in clinical trials for an antibody of the present invention if their genotype indicates that they are likely to respond significantly better to an antibody of the present invention as compared to one or more currently used antibody therapeutics.
- appropriate dosages and treatment regimens are determined using such genotype information.
- patients are selected for inclusion in a clinical trial or for receipt of therapy post-approval based on their polymorphism genotype, where such therapy contains an antibody engineered to be specifically efficacious for such population, or alternatively where such therapy contains an antibody that does not show differential activity to the different forms of the polymorphism.
- the present invention comprises diagnostic tests to identify patients who are likely to show a favorable clinical response to an antibody of the present invention, or who are likely to exhibit a significantly better response when treated with an antibody of the present invention versus one or more currently used antibody therapeutics. Any of a number of methods for determining Fc ⁇ R polymorphisms in humans known in the art may be used.
- the present invention comprises prognostic tests performed on clinical samples such as blood and tissue samples. Such tests may assay for effector function activity, including but not limited to ADCC, CDC, phagocytosis, and opsonization, or for killing, regardless of mechanism, of cancerous or otherwise pathogenic cells.
- ADCC assays are used to predict, for a specific patient, the efficacy of a given antibody of the present invention. Such information may be used to identify patients for inclusion or exclusion in clinical trials, or to inform decisions regarding appropriate dosages and treatment regimens. Such information may also be used to select a drug that contains a particular antibody that shows superior activity in such assay.
- Formulation [237] Pharmaceutical compositions are contemplated wherein an antibody of the present invention and one or more therapeutically active agents are formulated. Formulations of the antibodies of the present invention are prepared for storage by mixing the antibody having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A.
- Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, acetate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl orbenzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers
- the pharmaceutical composition that comprises the antibody of the present invention may be in a water-soluble form, such as being present as pharmaceutically acceptable salts, which is meant to include both acid and base addition salts.
- “Pharmaceutically acceptable acid addition salt” refers to those salts that retain the biological effectiveness of the free bases and that are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.
- inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like
- organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid,
- “Pharmaceutically acceptable base addition salts” include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly useful are the ammonium, potassium, sodium, calcium, and magnesium salts.
- Salts derived from pharmaceutically acceptable organic nontoxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine,. and ethanolamine.
- the formulations to be used for in vivo administration should be sterile. This is readily accomplished by filtration through sterile filtration membranes or other methods.
- a liposome is a small vesicle comprising various types of lipids, phospholipids and/or surfactant that is useful for delivery of a therapeutic agent to a mammal.
- Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., 1985, Proc Natl Acad Sci USA, 82:3688; Hwang et al., 1980, Proc Natl Acad Sci USA, 77:4030; US 4,485,045; US 4,544,545; and PCT WO 97/38731, each incorporated herein it its entirety by reference.
- Liposomes with enhanced circulation time are disclosed in US 5,013,556, incorporated herein it its entirety by reference.
- the components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.
- Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
- a chemotherapeutic agent or other therapeutically active agent is optionally contained within the liposome (Gabizon et al., 1989, J National Cancer Inst 81 :1484, incorporated herein it its entirety by reference).
- the antibody and other therapeutically active agents may also be entrapped in microcapsules prepared by methods including but not limited to coacervation techniques, interfacial polymerization (for example using hydroxymethylcellulose or gelatin- microcapsules, or poly-(methylmethacylate) microcapsules), colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules), and macroemulsions.
- sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymer, which matrices are in the form of shaped articles, e.g. films, or microcapsules.
- sustained-release matrices include polyesters, hydrogels (for example poly(2- hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (US 3,773,919, incorporated herein it its entirety by reference), copolymers of L-glutamic acid and gamma ethyl-L- glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the Lupron Depot® (which are injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), poly-D-(-)-3-hydroxybutyric acid, and ProLease® (commercially available from Alkermes), which is a microsphere-based delivery system composed of the desired bioactive molecule incorporated into a matrix of poly-DL-lactide-co-glycolide (PLG).
- PLA poly-DL-lactide-co-glycolide
- Administration of the pharmaceutical composition comprising an antibody of the present invention may be done in a variety of ways, including, but not limited to orally, subcutaneously, intravenously, intranasally, intraotically, transdermal ⁇ , topically (e.g., gels, salves, lotions, creams, etc.), intraperitoneally, intramuscularly, intrapulmonary, vaginally, parenterally, rectally, or intraocularly.
- the antibody may be directly applied as a solution or spray.
- the pharmaceutical composition may be formulated accordingly depending upon the manner of introduction.
- Subcutaneous administration may be desirable in some circumstances because the patient may self-administer the pharmaceutical composition.
- Many protein therapeutics are not sufficiently potent to allow for formulation of a therapeutically effective dose in the maximum acceptable volume for subcutaneous administration. This problem may be addressed in part by the use of protein formulations comprising arginine-HCI, histidine, and polysorbate (see WO 04091658, incorporated herein it its entirety by reference).
- Antibodies of the present invention may be more amenable to subcutaneous administration due to, for example, increased potency, improved serum half-life, or enhanced solubility.
- protein therapeutics are often delivered by IV infusion or bolus.
- the antibodies of the present invention may also be delivered using such methods. For example, administration may be by intravenous infusion with 0.9% sodium chloride as an infusion vehicle.
- Pulmonary delivery may be accomplished using an inhaler or nebulizer and a formulation comprising an aerosolizing agent.
- AERx® inhalable technology commercially available from Aradigm, or InhanceTM pulmonary delivery system commercially available from Nektar Therapeutics may be used.
- Antibodies of the present invention may be more amenable to intrapulmonary delivery.
- FcRn is present in the lung, and may promote transport from the lung to the bloodstream (e.g. Syntonix WO 04004798, Bitonti et al. (2004) Proc. Nat. Acad. Sci. 101 :9763-8, each incorporated herein it its entirety by reference).
- antibodies that bind FcRn more effectively in the lung or that are released more efficiently in the bloodstream may have improved bioavailability following intrapulmonary administration.
- Antibodies of the present invention may also be more amenable to intrapulmonary administration due to, for example, improved solubility or altered isoelectric point.
- antibodies of the present invention may be more amenable to oral delivery due to, for example, improved stability at gastric pH and increased resistance to proteolysis.
- FcRn appears to be expressed in the intestinal epithelia of adults (Dickinson et al. (1999) J. Clin. Invest. 104:903-11 , incorporated herein it its entirety by reference), so antibodies of the present invention with improved FcRn interaction profiles may show enhanced bioavailability following oral administration.
- FcRn mediated transport of antibodies may also occur at other mucus membranes such as those in the gastrointestinal, respiratory, and genital tracts (Yoshida et al. (2004) Immunity 20:769-83, incorporated herein it its entirety by reference).
- any of a number of delivery systems are known in the art and may be used to administer the antibodies of the present invention. Examples include, but are not limited to, encapsulation in liposomes, microparticles, microspheres (eg. PLA/PGA microspheres), and the like.
- an implant of a porous, non-porous, or gelatinous material, including membranes or fibers, may be used.
- controlled release systems may be used to release the antibody at or close to the desired location of action.
- the dosing amounts and frequencies of administration are, in one embodiment, selected to be therapeutically or prophylactically effective. As is known in the art, adjustments for protein degradation, systemic versus localized delivery, and rate of new protease synthesis, as well as the age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art.
- the concentration of the therapeutically active antibody in the formulation may vary from about 0.1 to 100 weight %. In one embodiment, the concentration of the antibody is in the range of 0.003 ⁇ M to 1.0 molar. In order to treat a patient, a therapeutically effective dose of the antibody of the present invention may be administered.
- terapéuticaally effective dose herein is meant a dose that produces the effects for which it is administered.
- the exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. Dosages may range from 0.0001 to 100 mg/kg of body weight or greater, for example 0.1 , 1 , 10, or 50 mg/kg of body weight, e.g., 1 to 10mg/kg of body weight. [250] In some embodiments, only a single dose of the antibody is used. In other embodiments, multiple doses of the antibody are administered.
- the elapsed time between administrations may be less than 1 hour, about 1 hour, about 1-2 hours, about 2-3 hours, about 3-4 hours, about 6 hours, about 12 hours, about 24 hours, about 48 hours, about 2-4 days, about 4-6 days, about 1 week, about 2 weeks, or more than 2 weeks.
- the antibodies of the present invention are administered in metronomic dosing regimes, either by continuous infusion or frequent administration without extended rest periods. Such metronomic administration may involve dosing at constant intervals without rest periods. Typically such regimens encompass chronic low-dose or continuous infusion for an extended period of time, for example 1-2 days, 1-2 weeks, 1-2 months, or up to 6 months or more. The use of lower doses may minimize side effects and the need for rest periods.
- the antibody of the present invention and one or more other prophylactic or therapeutic agents are cyclically administered to the patient. Cycling therapy involves administration of a first agent at one time, a second agent at a second time, optionally additional agents at additional times, optionally a rest period, and then repeating this sequence of administration one or more times. The number of cycles is typically from 2 - 10. Cycling therapy may reduce the development of resistance to one or more agents, may minimize side effects, or may improve treatment efficacy. [253] Combination therapies [254]
- the antibodies of the present invention may be administered concomitantly with one or more other therapeutic regimens or agents. The additional therapeutic regimes or agents may be used to improve the efficacy or safety of the antibody.
- an antibody of the present invention may be administered to the patient along with chemotherapy, radiation therapy, or both chemotherapy and radiation therapy.
- the antibody of the present invention may be administered in combination with one or more other prophylactic or therapeutic agents, including but not limited to cytotoxic agents, chemotherapeutic agents, cytokines, growth
- R3 inhibitory agents anti-hormonal agents, kinase inhibitors, anti-angiogenic agents, cardioprotectants, immunostimulatory agents, immunosuppressive agents, agents that promote proliferation of hematological cells, angiogenesis inhibitors, protein tyrosine kinase (PTK) inhibitors, additional antibodies, Fc ⁇ Rllb or other Fc receptor inhibitors, or other therapeutic agents.
- the terms "in combination with” and “co-administration” are not limited to the administration of the prophylactic or therapeutic agents at exactly the same time. Instead, it is meant that the antibody of the present invention and the other agent or agents are administered in a sequence and within a time interval such that they may act together to provide a benefit that is increased versus treatment with only either the antibody of the present invention or the other agent or agents. In one embodiment, that the antibody and the other agent or agents act additively, e.g., they act synergistically. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
- the antibodies of the present invention are administered with one or more additional molecules comprising antibodies or Fc.
- the antibodies of the present invention may be co-administered with one or more other antibodies that have efficacy in treating the same disease or an additional comorbidity; for example two antibodies may be administered that recognize two antigens that are overexpressed in a given type of cancer, or two antigens that mediate pathogenesis of an autoimmune or infectious disease.
- anti-cancer antibodies examples include, but are not limited to, anti-17-1A cell surface antigen antibodies such as PanorexTM (edrecolomab); anti- 4-1BB antibodies; anti-4Dc antibodies; anti-A33 antibodies such as A33 and CDP-833; anti- ⁇ 4 ⁇ 1 integrin antibodies such as natalizumab; anti- ⁇ 4 ⁇ 7 integrin antibodies such as LDP-02; anti- ⁇ V ⁇ 1 integrin antibodies such as F-200, M-200, and SJ-749; anti- ⁇ V ⁇ 3 integrin antibodies such as abciximab, CNTO-95, Mab-17E6, and VitaxinTM; anti-complement factor 5 (C5) antibodies such as 5G1.1 ; anti-CA125 antibodies such as OvaRex® (oregovomab); anti-CD3 antibodies such as Nuvion® (visilizumab) and Rexomab; anti-CD4 antibodies such as IDEC-151
- anti-complement factor 5 (C5) antibodies such as 5
- anti-CD23 antibodies such as IDEC-152
- anti-CD25 antibodies such as basiliximab and Zenapax® (daclizumab)
- anti-CD30 antibodies such as AC10, MDX-060, and SGN-30
- anti-CD33 antibodies such as Mylotarg® (gemtuzumab ozogamicin), Oncolysin M, and Smart M195
- anti-CD38 antibodies anti-CD40 antibodies such as SGN-40 and toralizumab
- anti-CD40L antibodies such as 5c8, AntovaTM, and IDEC- 131
- anti-CD44 antibodies such as bivatuzumab
- anti-CD46 antibodies anti-CD402 antibodies such as Campath® (alemtuzumab); anti-CD405 antibodies such as SC-1 ; anti- CD406 antibodies such as huN901-DM1; anti-CD64 antibodies such as MDX-33; anti- CD66e antibodies such as
- anti-HLA antibodies such as Oncolym®, Smart 1 D10
- anti-HM1.24 antibodies anti-ICAM antibodies such as ICM3
- anti-lgA receptor antibodies anti-IGF-1 antibodies
- anti-IGF-1 R antibodies such as IMC-A12
- anti-IL-6 antibodies such as CNTO-328 and elsilimomab
- anti-IL-15 antibodies such as HuMaxTM-IL15
- anti-KDR antibodies anti-laminin 5 antibodies
- anti-Lewis Y antigen antibodies such as Hu3S193 and IGN-311
- anti-MCAM antibodies anti-Mud antibodies such as BravaRex and TriAb
- anti-NCAM antibodies such as ERIC-1 and ICRT
- anti-PEM antigen antibodies such as Theragyn and Therex
- anti-PSA antibodies anti-PSCA antibodies such as IG8
- anti-Ptk antibodies anti-PT
- Anti-STEAP antibodies such as CC49-SCA and MDX-220
- anti-TGF- ⁇ antibodies such as CAT-152
- anti-TNF- ⁇ antibodies such as CDP571 , CDP870, D2E7, Humira® (adalimumab), and Remicade® (infliximab)
- anti-TRAIL-R1 and TRAIL-R2 antibodies anti-VE-cadherin-2 antibodies
- anti-VLA-4 antibodies such as AntegrenTM.
- anti-idiotype antibodies including but not limited to the GD3 epitope antibody BEC2 and the gp72 epitope antibody 105AD7
- bispecific antibodies including but not limited to the anti-CD3/CD20 antibody Bi20 may be used.
- antibodies that may be co-administered to treat autoimmune or inflammatory disease, transplant rejection, GVHD, and the like include, but are not limited to, anti- ⁇ 4 ⁇ 7 integrin antibodies such as LDP-02, anti-beta2 integrin antibodies such as LDP- 01 , anti-complement (C5) antibodies such as 5G1.1 , anti-CD2 antibodies such as BTI-322, MEDI-507, anti-CD3 antibodies such as OKT3, SMART anti-CD3, anti-CD4 antibodies such as IDEC-151 , MDX-CD4, OKT4A, anti-CD11a antibodies, anti-CD14 antibodies such as IC14, anti-CD18 antibodies, anti-CD23 antibodies such as IDEC 152, anti-CD25 antibodies such as Zenapax, anti-CD40L antibodies such as 5c8, Antova, IDEC-131 , anti-CD64 antibodies such as MDX-33, anti-CD80 antibodies such as IDEC-114, anti-CD147 antibodies such as ABX-C
- Fc-containing molecules that may be coadministered to treat autoimmune or inflammatory disease, transplant rejection, GVHD, and the like include, but are not limited to, the p75 TNF receptor/Fc fusion Enbrel® (etanercept) and Regeneron's IL-1 trap.
- antibodies that may be co-administered to treat infectious diseases include, but are not limited to, anti-anthrax antibodies such as ABthrax, anti-CMV antibodies such as CytoGam and sevirumab, anti-cryptosporidium antibodies such as CryptoGAM, Sporidin-G, anti-helicobacter antibodies such as Pyloran, anti-hepatitis B antibodies such as HepeX-B, Nabi-HB, anti-HIV antibodies such as HRG-214, anti-RSV antibodies such as felvizumab, HNK-20, palivizumab, RespiGam, and anti-staphylococcus antibodies such as Aurexis, Aurograb, BSYX-A110, and SE-Mab.
- anti-anthrax antibodies such as ABthrax
- anti-CMV antibodies such as CytoGam and sevirumab
- anti-cryptosporidium antibodies such as CryptoGAM
- Sporidin-G anti-helicobacter antibodies
- anti-helicobacter antibodies such as Py
- the antibodies of the present invention may be co-administered or with one or more other molecules that compete for binding to one or more Fc receptors.
- co-administering inhibitors of the inhibitory receptor Fc ⁇ Rllb may result in increased effector function.
- co-administering inhibitors of the activating receptors such as Fc ⁇ Rllla may minimize unwanted effector function.
- Fc receptor inhibitors include, but are not limited to, Fc molecules that are engineered to act as competitive inhibitors for binding to Fc ⁇ Rllb, Fc ⁇ Rllla, or other Fc receptors, as well as other immunoglobulins and specifically the treatment called IVIg (intravenous immunoglobulin).
- the inhibitor is administered and allowed to act before the antibody is administered.
- An alternative way of achieving the effect of sequential dosing would be to provide an immediate release dosage form of the Fc receptor inhibitor and then a sustained release formulation of the antibody of the invention.
- the immediate release and controlled release formulations could be administered separately or be combined into one unit dosage form.
- Administration of an Fc ⁇ Rllb inhibitor may also be used to limit unwanted immune responses, for example anti-Factor VIII antibody response following Factor VIII administration to hemophiliacs.
- the antibodies of the present invention are administered with a chemotherapeutic agent.
- chemotherapeutic agent as used herein is meant a chemical compound useful in the treatment of cancer.
- examples of chemotherapeutic agents include but are not limited to alkylating agents such as thiotepa and cyclosphosphamide
- CYTOXANTM alkyl sulfonates such as busulfan, improsulfan and piposulfan
- androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone
- anti-adrenals such as aminoglutethimide, mitotane, trilostane
- anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin
- antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, dox
- paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ.) and docetaxel (TAXOTERE®, Rhne-Poulenc Rorer, Antony, France); topoisomerase inhibitor RFS 2000; thymidylate synthase inhibitor (such as Tomudex); additional chemotherapeutics including aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; difluoromethylornithine (DMFO); elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; Ionidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarub
- a chemotherapeutic or other cytotoxic agent may be administered as a prodrug.
- prodrug as used herein is meant a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form.
- the prodrugs that may find use with the present invention include but are not limited to phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, beta-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs which can be converted into the more active cytotoxic free drug.
- a variety of other therapeutic agents may find use for administration with the antibodies of the present invention.
- the antibody is administered with an anti-angiogenic agent.
- anti-angiogenic agent as used herein is meant a compound that blocks, or interferes to some degree, the development of blood vessels.
- the anti-angiogenic factor may, for instance, be a small molecule or a protein, for example an antibody, Fc fusion, or cytokine, that binds to a growth factor or growth factor receptor involved in promoting angiogenesis.
- one such anti-angiogenic factor herein is an antibody that binds to Vascular Endothelial Growth Factor (VEGF).
- VEGF Vascular Endothelial Growth Factor
- Other agents that inhibit signaling through VEGF may also be used, for example RNA-based therapeutics that reduce levels of VEGF or VEGF-R expression, VEGF-toxin fusions, Regeneron's VEGF-trap, and antibodies that bind VEGF-R.
- the antibody is administered with a therapeutic agent that induces or enhances adaptive immune response, for example an antibody that targets CTLA-4.
- Additional anti-angiogenesis agents include, but are not limited to, angiostatin (plasminogen fragment), antithrombin III, angiozyme, ABT-627, Bay 12-9566, benefin, bevacizumab, bisphosphonates, BMS-275291 , cartilage-derived inhibitor (CDI), CAI, CD409 complement fragment, CEP-7055, CoI 3, combretastatin A-4, endostatin (collagen XVIII fragment), farnesyl transferase inhibitors, fibronectin fragment, gro-beta, halofuginone, heparinases, heparin hexasaccharide fragment, HMV833, human chorionic gonadotropin (hCG), IM-862, interferon alpha, interferon beta, interferon gamma, interferon inducible protein 10 (IP-10), interleukin-12, kringle 5 (plasminogen fragment), marimastat, metalloproteinas
- TIMPs 2-methodyestradiol
- MMI 270 CCS 27023A
- plasminogen activator inhibitor PAI
- platelet factor-4 PF4
- prinomastat prolactin 16kDa fragment
- proliferin-related protein PRP
- PTK 787/ZK 222594 retinoids
- solimastat squalamine
- SS3304 SU5416
- SU6668 SU11248, tetrahydrocortisol-S, tetrathiomolybdate
- thalidomide thrombospondin-1
- TNP-1 TNP-470
- TGF- ⁇ transforming growth factor beta
- vasculostatin vasostatin (calreticulin fragment)
- ZS6126 and ZD6474.
- the antibody is administered with a tyrosine kinase inhibitor.
- tyrosine kinase inhibitor as used herein is meant a molecule that inhibits to some extent tyrosine kinase activity of a tyrosine kinase.
- inhibitors include but are not limited to quinazolines, such as PD 153035, 4-(3-chloroanilino) quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines, such as CGP 59326, CGP 60261 and CGP 62706; pyrazolopyrimidines, 4-(phenylamino)-7H-pyrrolo(2,3-d) pyrimidines; curcumin (diferuloyl methane, 4,5-bis (4-fluoroanilino)phthalimide); tyrphostines containing nitrothiophene moieties; PD-0183805 (Warner-Lambert); antisense molecules (e.g.
- the antibody is administered with one or more immunomodulatory agents.
- immunomodulatory agents may increase or decrease production of one or more cytokines, up- or down-regulate self-antigen presentation, mask MHC antigens, or promote the proliferation, differentiation, migration, or activation state of one or more types of immune cells.
- Immunomodulatory agents include but are not limited to: non-steroidal antiinflammatory drugs (NSAIDs) such as aspirin, ibuprofen, celecoxib, diclofenac, etodolac, fenoprofen, indomethacin, ketoralac, oxaprozin, nabumentone, sulindac, tolmentin, rofecoxib, naproxen, ketoprofen, and nabumetone; steroids (eg.
- NSAIDs non-steroidal antiinflammatory drugs
- glucocorticoids dexamethasone, cortisone, hydroxycortisone, methylprednisolone, prednisone, prednisolone, trimcinolone, azulfidineicosanoids such as prostaglandins, thromboxanes, and leukotrienes; as well as topical steroids such as anthralin, calcipotriene, clobetasol, and tazarotene); cytokines such as TGFb, IFNa, IFNb, IFNg, IL-2, IL-4, IL-10; cytokine, chemokine, or receptor antagonists including antibodies, soluble receptors, and receptor-Fc fusions against BAFF, B7, CCR2, CCR5, CD2, CD3, CD4, CD6, CD7, CD8, CD11 , CD14, CD15, CD17, CD18, CD20, CD23, CD28, CD40, CD40L, CD44, CD45, CD402, CD64,
- TNF ⁇ , TNF-R1 , T-cell receptor including Enbrel® (etanercept), Humira® (adalimumab), and Remicade® (infliximab); heterologous anti-lymphocyte globulin; other immunomodulatory molecules such as 2-amino-6-aryl-5 substituted pyrimidines, anti-idiotypic antibodies for MHC binding peptides and MHC fragments, azathioprine, brequinar, bromocryptine, cyclophosphamide, cyclosporine A, D-penicillamine, deoxyspergualin, FK506, glutaraldehyde, gold, hydroxychloroquine, leflunomide, malononitriloamides (e.g.
- antibodies of the present invention are administered with a cytokine.
- cytokine as used herein is meant a generic term for proteins released by one cell population that act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones.
- cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor- alpha and -beta; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-beta; platelet-growth factor; transforming growth factors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growth factor-l and -II; erythropoietin (E
- cytokines or other agents that stimulate cells of the immune system are co-administered with the antibody of the present invention.
- a mode of treatment may enhance desired effector function.
- agents that stimulate NK cells including but not limited to IL-2 may be co-administered.
- agents that stimulate macrophages including but not limited to C5a, formyl peptides such as N-formyl-methionyl-leucyl-phenylalanine (Beigier-Bompadre et al. (2003) Scand. J. Immunol. 57: 221-8, incorporated herein it its entirety by reference), may be co-administered.
- agents that stimulate neutrophils including but not limited to G-CSF, GM-CSF, and the like may be administered.
- agents that promote migration of such immunostimulatory cytokines may be used.
- additional agents including but not limited to interferon gamma, IL-3 and IL-7 may promote one or more effector functions.
- cytokines or other agents that inhibit effector cell function are co-administered with the antibody of the present invention. Such a mode of treatment may limit unwanted effector function.
- the antibody is administered with one or more antibiotics, including but not limited to: aminoglycoside antibiotics (eg. apramycin, arbekacin, bambermycins, butirosin, dibekacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, ribostamycin, sisomycin, spectrinomycin), aminocyclitols (eg. sprctinomycin), amphenicol antibiotics (eg. azidamfenicol, chloramphenicol, florfrnicol, and thiamphemicol), ansamycin antibiotics (eg.
- aminoglycoside antibiotics eg. apramycin, arbekacin, bambermycins, butirosin, dibekacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, ribostamycin, sisomycin, spect
- rifamide and rifampin carbapenems (eg. imipenem, meropenem, panipenem); cephalosporins (eg. cefaclor, cefadroxil, cefamandole, cefatrizine, cefazedone, cefozopran, cefpimizole, cefpiramide, cefpirome, cefprozil, cefuroxine, cefixime, cephalexin, cephradine ), cephamycins (cefbuperazone, cefoxitin, cefminox, cefmetazole, and cefotetan); lincosamides (eg.
- clindamycin, lincomycin macrolide (eg. azithromycin, brefeldin A, clarithromycin, erythromycin, roxithromycin, tobramycin), monobactams (eg. aztreonam, carumonam, and tigemonam); mupirocin; oxacephems (eg. flomoxef, latamoxef, and moxalactam); penicillins (eg.
- bacitracin colistin, polymixin B, teicoplanin, vancomycin
- quinolones amifloxacin, cinoxacin, ciprofloxacin, enoxacin, enrofloxacin, feroxacin, flumequine, gatifloxacin, gemifloxacin, grepafloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, oxolinic acid, pefloxacin, pipemidic acid, rosoxacin, rufloxacin, sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin); rifampin; streptogramins (eg.
- quinupristin, dalfopristin quinupristin, dalfopristin
- sulfonamides sulfanilamide, sulfamethoxazole
- tetracyclenes chlortetracycline, demeclocycline hydrochloride, demethylchlortetracycline, doxycycline, duramycin, minocycline, neomycin, oxytetracycline, streptomycin, tetracycline, vancomycin).
- Anti-fungal agents such as amphotericin B, ciclopirox, clotrimazole, econazole, fluconazole, flucytosine, itraconazole, ketoconazole, niconazole, nystatin, terbinafine, terconazole, and tioconazole may also be used.
- Antiviral agents including protease inhibitors, reverse transcriptase inhibitors, and others, including type I interferons, viral fusion inhibitors, and neuramidase inhibitors, may also be used.
- antiviral agents include, but are not limited to, acyclovir, adefovir, amantadine, amprenavir, clevadine, enfuvirtide, entecavir, foscarnet, gangcyclovir, idoxuridine, indinavir, lopinavir, pleconaril, ribavirin, rimantadine, ritonavir, saquinavir, trifluridine, vidarabine, and zidovudine.
- the antibodies of the present invention may be combined with other therapeutic regimens.
- the patient to be treated with an antibody of the present invention may also receive radiation therapy.
- Radiation therapy can be administered according to protocols commonly employed in the art and known to the skilled artisan. Such therapy includes but is not limited to cesium, iridium, iodine, or cobalt radiation.
- the radiation therapy may be whole body irradiation, or may be directed locally to a specific site or tissue in or on the body, such as the lung, bladder, or prostate.
- radiation therapy is administered in pulses over a period of time from about 1 to 2 weeks. The radiation therapy may, however, be administered over longer periods of time.
- radiation therapy may be administered to patients having head and neck cancer for about 6 to about 7 weeks.
- the radiation therapy may be administered as a single dose or as multiple, sequential doses.
- the skilled medical practitioner can determine empirically the appropriate dose or doses of radiation therapy useful herein.
- the antibody of the present invention and one or more other anti-cancer therapies are employed to treat cancer cells ex vivo. It is contemplated that such ex vivo treatment may be useful in bone marrow transplantation and particularly, autologous bone marrow transplantation.
- treatment of cells or tissue(s) containing cancer cells with antibody and one or more other anti-cancer therapies, such as described above can be employed to deplete or substantially deplete the cancer cells prior to transplantation in a recipient patient.
- the antibodies of the invention may employ in combination with still other therapeutic techniques such as surgery or phototherapy.
- Example 1 Anti-CD40 antibodies with amino acid modifications that enhance effector function
- the anti-CD40 antibodies of the invention are intended as clinical candidates for anti- cancer therapeutics.
- variant versions of anti-CD40 antibodies were engineered.
- Figure 5 provides some heavy and light chain variable region sequences of the anti- CD40 antibodies S2C6 (Paulie S. et al., 1984. Cancer Immunol Immunother. 17:173-179), G28-5 (Clark E. A. et al., 1988. Eur J Immunol. 18:451-457), and 5D12 (de Boer M. et al., 1992. J Immunol Methods.
- HO and LO The mouse, parent chimeric heavy and light chains are labeled HO and LO, respectively.
- Variant S239D/I332E effector function enhanced anti-CD40 was constructed in the Fc region of a hybrid lgG1/lgG2 ( Figure 1) antibody in the pcDNA3.1Zeo vector using QuikChange mutagenesis techniques (Stratagene). All sequences were sequenced to confirm the fidelity of the sequence. Plasmids containing heavy chain gene (VH-CH 1-CH2-CH3) (wild-type or variants) were co- transfected with plasmid containing light chain gene (VL-CLK) into 293T cells. Media were harvested 5 days after transfection, and antibodies were purified from the supernatant using protein A affinity chromatography (Pierce, Catalog # 20334).
- PBMCs Human peripheral blood monocytes
- CD40 positive cancer cells were used as target cells.
- Target cells were seeded at 20,000 cells/well and treated with designated antibodies in triplicates.
- PBMCs isolated using a Ficoll gradient were added in excess to target cells and co-cultured for 4 hrs before processing for LDH activity using the Cytotoxicity Detection Kit according to the manufacturer's instructions.
- Figure 6 shows the results of ADCC assays comparing WT IgGI , effector function enhanced anti-CD40 antibodies (hybrid S239D/I332E), and/or effector function enhanced rituximab (hybrid S239D/I332E) on the cell lines Daudi (Burkitt's Lymphoma), Raji (Burkitt's Lymphoma), and RPMI8226 (Multiple Myeloma) for antibodies S2C6 ( Figure 6a), 5D12 ( Figure 6b), and G28-5 ( Figure 6c).
- the graphs show that the antibodies differ not only in their EC50, reflecting their relative potency, but also in the maximal level of ADCC attainable by the antibodies at saturating concentrations, reflecting their relative efficacy. These two terms, potency and efficacy, are sometimes used loosely to refer to desired clinical properties. In the current experimental context, however, they are denoted as specific quantities, and therefore are here explicitly defined.
- potency as used in the current experimental context is meant the EC50 of an antibody.
- efficacy as used in the current experimental context is meant the maximal possible effector function of an antibody at saturating levels.
- Considerable enhancements in potency and efficacy are observed for the Fc variant antibodies with enhanced effector function as compared to the antibodies with WT Fc region. All three effector function enhanced antibodies displayed ADCC on the Multiple Myeloma cell line RPMI8226, with S2C6 showing the most efficacy, indicating that this antibody may be an effective treatment option for Multiple Myeloma.
- Example 2 Platelet activation assay with anti-CD40 antibody S2C6
- a platelet activation assay was performed. Platelets were obtained from fresh blood drawn by venipuncture in NaCitrate and spun at low speed (100xg) for 15 minutes from which the straw colored upper phase was removed as platelet rich plasma (PRP). The PRP was further spun at 700xg for 2.5 minutes and the pellet was saved and washed twice with 10 mM EDTA, 1% FBS in CaCI 2 and MgCI 2 free PBS. The platelet pellet was resuspended in EDTA/FBS/PBS at 1.5x of the PRP volume and used in the experiment. Antibody serial
- the cross-linking antibodies used were goat anti-human IgG Fc specific biotinylated antibody and goat anti-mouse IgG Fc specific antibody for human or mouse antibodies, respectively.
- HS-Sultan cells were pelleted and resuspended in 2%FBS/RPMI1640 with HEPES and NaPyruvate at 100,000 cells/ml and 50 ⁇ l was added to the prepared antibody dilutions. After 4 days at 37°C, the amount of ATP present in the live cells was detected using the ATP dependent luminescent viability assay kit (Cell TiterGlo, Promega Corp.). Human IgG with and without CD40 ligand and cells only were used as controls. Both S2C6 and 5D12 show anti-proliferative effects on the HS-Sultan cell line.
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US88629107P | 2007-01-23 | 2007-01-23 | |
PCT/US2008/051812 WO2008091954A2 (en) | 2007-01-23 | 2008-01-23 | Optimized cd40 antibodies and methods of using the same |
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CN109265552A (zh) * | 2012-10-30 | 2019-01-25 | 埃派斯进有限公司 | 抗-cd40抗体及其使用方法 |
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AU2006249305B2 (en) * | 2005-05-26 | 2012-10-18 | Genentech, Inc. | Humanized anti-CD40 antibodies and their methods of use |
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